Antiviral oligonucleotides targeting HSV and CMV

ABSTRACT

Random sequence oligonucleotides that have antiviral activity are described, along with their use as antiviral agents. In many cases, the oligonucleotides are greater than 40 nucleotides in length. Also described are methods for the prophylaxis or treatment of a viral infection in a human or animal, and a method for the prophylaxis treatment of cancer caused by oncoviruses in a human or animal. The methods typically involve administering to a human or animal in need of such treatment, a pharmacologically acceptable, therapeutically effective amount of at least oligonucleotide that does not act by a sequence complementary mode of action.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of Juteau & Vaillant,PCT application serial number not yet assigned, filed Sep. 11, 2003,entitled ANTIVIRAL OLIGONUCLEOTIDES, and claims the benefit of Vaillant& Juteau, U.S. Provisional Appl. 60/430,934, filed Dec. 5, 2002 and ofVaillant & Juteau, U.S. Provisional Appl. 60/410,264, filed Sep. 13,2002, all of which are incorporated herein by reference in theirentireties, including drawings.

FIELD OF THE INVENTION

[0002] The present invention relates to oligonucleotides havingantiviral activities and their use as therapeutic agents in viralinfections caused by human and animal viruses and in cancers caused byoncogene viruses and in other diseases whose etiology is viral-based.

BACKGROUND OF THE INVENTION

[0003] The following discussion is provided solely to assist theunderstanding of the reader, and does not constitute an admission thatany of the information discussed or references cited constitute priorart to the present invention.

[0004] Many important infectious diseases afflicting mankind are causedby viruses. Many of these diseases, including rabies, smallpox,poliomyelitis, hepatitis, yellow fever, immune deficiencies and variousencephalitic diseases, are frequently fatal. Others are significant inthat they are highly contagious and create acute discomfort such asinfluenza, measles, mumps and chickenpox, as well as respiratory orgastrointestinal disorders. Others such as rubella and cytomegaloviruscan cause congenital abnormalities. Finally there are viruses, known asoncoviruses, which can cause cancer in humans and animals.

[0005] Among viruses, the family of Herpesviridae is of great interest.The Herpesviridae are a ubiquitous class of icoshedral, double strandedDNA viruses. Of over 100 characterized members of Herpesviridae (HHV),only eight infect humans. The best known among these are Herpes simplextype 1 (HSV-1), Herpes simplex type 2 (HSV-2), Varicella zoster (chickenpox or shingles), cytomegalovirus (CMV) and Epstein-Barr virus (EBV).The prevalence of Herpes viruses in humans is high, affecting at leastone third of the worldwide population; and in the United States, 70-80%of the population have some kind of Herpes infection. While thepathology of Herpes infections are usually not dangerous, as in the caseof HSV-1 which usually only causes short lived lesions around the mouthand face, these viruses are also known to be the cause of more dangeroussymptoms, which vary from genital ulcers and discharge to fetalinfections which can lead to encephalitis (15% mortality) ordisseminated infection (40% mortality).

[0006] Herpes viruses are highly disseminated in nature and highlypathogenic for man. For example, Epstein-Barr virus (EBV) is known tocause infectious mononucleosis in late childhood or adolescence or inyoung adults. The hallmarks of acute infectious mononucleosis are sorethroat, fever, headache, lymphadenopathy, enlarged tonsils and atypical, dividing lymphocytes in the peripheral blood. Othermanifestations frequently include mild hepatitis, splenomegaly andencephalitis. EBV is also associated with two forms of cancer: Burkitt'slymphoma (BL) and the nasopharyngeal carcinoma (NPC). In endemic areasof equatorial Africa, BL is the most common childhood malignancy,accounting for approximately 80% of cancers in children. Whilemoderately observed in North American Caucasians, NPC is one of the mostcommon cancers in Southern China with age incidence of 25 to 55 years.EBV, like the cytomegalovirus, is also associated with post-transplantlymphoproliferative disease, which is a potentially fatal complicationof chronic immunosuppression following solid organ or bone marrowtransplantation.

[0007] Other diseases are also associated with HSV, including skin andeye infections, for example, chorioretinitis or keratoconjunctivitis.Approximately 300,000 cases of HSV infections of the eye are diagnosedyearly in the United States.

[0008] AIDS (acquired immunodeficiency syndrome) is caused by the humanimmunodeficiency virus (HIV). By killing or damaging cells of the body'simmune system, HIV progressively destroys the body's ability to fightinfections and certain cancers. There are currently approximately 42million people living with HIV/AIDS worldwide. A total of 3.1 millionpeople died of HIV/AIDS related causes in 2002. The ultimate goal ofanti-HIV drug therapy is to prevent the virus from reproducing anddamaging the immune system. Although substantial progress has been madeover the past fifteen years in the fight against HIV, a cure stilleludes medical science. Today, physicians have more than a dozenantiretroviral agents in three different drug classes to manage thedisease. Typically, drugs from two or three classes are prescribed in avariety of combinations known as HAART (Highly Active AntiRetroviralTreatment). HAART therapies typically comprise two nucleoside reversetranscriptase inhibitors drugs with a third drug, either a proteaseinhibitor or a non-nucleoside reverse transcriptase inhibitor. Clinicalstudies have shown that HAART is the most effective means of reducingviral loads and minimizing the likelihood of drug resistance.

[0009] While HAART has been shown to reduce the amount of HIV in thebody, commonly known as viral load, tens of thousands of patientsencounter significant problems with this therapy. Some side effects areserious and include abnormal fat metabolism, kidney stones, and heartdisease. Other side effects such as nausea, vomiting, and insomnia areless serious, but still problematic for HIV patients that need chronicdrug therapy for a lifetime.

[0010] Currently approved anti-HIV drugs work by entering an HIVinfected CD4+ T cell and blocking the function of a viral enzyme, eitherthe reverse transcriptase or a protease. HIV needs both of these enzymesin order to reproduce. However, HIV frequently mutates and becomeresistant, rendering reverse transcriptase or protease inhibitor drugsineffective. Once resistance occurs, viral loads increase and dictatethe need to switch the ineffective agent for another antiretroviralagent. Unfortunately, when a virus becomes resistant to one drug in aclass, other drugs in that class may become less effective. Thisphenomenon known as cross-resistance, occurs because many anti-HIV drugswork in similar manners. The occurrence of drug cross-resistance ishighly undesirable because it reduces the available number of treatmentoptions for patients.

[0011] There is therefore a great need for the development of otherantiviral agents effective against HIV that work through othermechanisms of action against which the virus has not developedresistance. This is becoming especially important in view of recent datashowing that 1 out of 10 patients newly diagnosed with HIV in Europe, isinfected with a strain of HIV already resistant to at least one of theapproved drug on the market.

[0012] Respiratory syncytial virus (RSV) causes upper and lowerrespiratory tract infections. It is a negative-sense, enveloped RNAvirus and is highly infectious. It commonly affects young children andis the most common cause of lower respiratory tract illness in infants.RSV infections are usually associated with moderate-to-severe cold-likesymptoms. However, severe lower respiratory tract disease may occur atany age, especially in elderly or immunocompromised patients. ChildrenWith severe infections may require oxygen therapy and, in certain cases,mechanical ventilation. According to the American Medical Association,an increasing number of children are being hospitalized forbronchiolitis, often caused by RSV infection. RSV infections alsoaccount for approximately one-third of community-associated respiratoryvirus infections in patients in bone marrow transplant centers. In theelderly population, RSV infection has been recently recognized to bevery similar in severity to influenza virus infection.

[0013] Influenza (INF), also known as the flu, is a contagious diseasethat is caused by the influenza virus. It attacks the respiratory tractin humans (nose, throat, and lungs). An average of about 36,000 peopleper year in the United States die from influenza, and 114,000 per yearrequire hospitalization as a result of influenza.

[0014] In all infectious diseases, the efficacy of a given therapy oftendepends on the host immune response. This is particularly true forherpes viruses, where the ability of all herpes viruses to establishlatent infections results in an extremely high incidence of reactivatedinfections in immunocompromised patients. In renal transplantrecipients, 40% to 70% reactivate latent HSV infections, and 80% to 100%reactivate CMV infections. Such viral reactivations have also beenobserved with AIDS patients.

[0015] The hepatitis B virus (HBV) is a DNA virus that belongs to theHepadnaviridae family of viruses. HBV causes hepatitis B in humans. Itis estimated that 2 billion people have been infected (1 out of 3people) in the world. About 350 million people remain chronicallyinfected and an estimated 1 million people die each year from hepatitisB and its complications. HBV can cause lifelong infection, cirrhosis ofthe liver, liver cancer, liver failure, and death. The virus istransmitted through blood and bodily fluids. This can occur throughdirect blood-to-blood contact, unprotected sex, use of unsterileneedles, and from an infected woman to her newborn during the deliveryprocess. Most healthy adults (90%) who are infected will recover anddevelop protective antibodies against future hepatitis B infections. Asmall number (5-10%) will be unable to get rid of the virus and willdevelop chronic infections while 90% of infants and up to 50% of youngchildren develop chronic infections when infected with the virus.Alpha-interferon is the most frequent type of treatment used.Significant side effects are related to this treatment includingflu-like symptoms, depression, rashes, other reactions and abnormalblood counts. Another treatment option includes 3TC which also has manyside effects associated with its use. In the last few years, there hasbeen an increasing number of reports showing that patients treated with3TC are developing resistant strains of HBV. This is especiallyproblematic in the population of patients who are co-infected with HBVand HIV. There is clearly an urgent need to develop new antiviraltherapies against this virus.

[0016] Hepatitis C virus (HCV) infection is the most common chronicbloodborne infection in the United States where the number of infectedpatients likely exceeds 4 million. This common viral infection is aleading cause of cirrhosis and liver cancer, and is now the leadingreason for liver transplantation in the United States. Recovery frominfection is uncommon, and about 85 percent of infected patients becomechronic carriers of the virus and 10 to 20 percent develop cirrhosis. Itis estimated that there are currently 170 million people worldwide whoare chronic carriers. According to the Centers for Disease Control andPrevention, chronic hepatitis C causes between 8,000 and 10,000 deathsand leads to about 1,000 liver transplants in the United States aloneeach year. There is no vaccine available for hepatitis C. Prolongedtherapy with interferon alpha, or the combination of interferon withRibavirin, is effective in only about 40 percent of patients and causessignificant side effects.

[0017] Today, the therapeutic outlook for viral infections in general isnot favourable. In general, therapies for viruses have mediocreefficacies and are associated with strong side effects which eitherprevent the administration of an effective dosage or prevent long termtreatment. Three clinical situations which exemplify these problems areherpesviridae, HIV and RSV infections.

[0018] In the case of herpesviridae, there are five major treatmentscurrently approved for use in the clinic: idoxuridine, vidarabine,acyclovir, foscarnet and ganciclovir. While having limited efficacy,these treatments are also fraught with side effects. Allergic reactionshave been reported in 35% of patients treated with idoxuridine,vidarabine can result in gastrointestional disturbances in 15% ofpatients and acyclovir, foscarnet and ganciclovir, being nucleosideanalogs, affect DNA replication in host cells. In the case ofganciclovir, neutropenia and thrombocytopenia are reported in 40% ofAIDS patients treated with this drug.

[0019] While there are many different drugs currently available for thetreatment of HIV infections, all of these are associated with sideeffects potent enough to require extensive supplemental medication togive patients a reasonable quality of life. The additional problem ofdrug resistant strains of HIV (a problem also found in herpesviridaeinfections) usually requires periodic changing of the treatment cocktailand in some cases, makes the infection extremely difficult to treat.

[0020] The treatment of RSV infections in young infants is anotherexample of the urgent need for new drug development. In this case, theusual line of treatment is to deliver Ribavirin by inhalation using asmall-particule aerosol in an isolation tent. Not only is Ribavirin onlymildly effective, but its uses is associated with significant sideeffects. In addition, the potential release of the drug has caused greatconcern in hospital personnel because of the known teratogenicity ofRibavirin.

[0021] It is clear that for any new emerging antiviral drug beingdeveloped, it would be highly desirable to incorporate the threefollowing features: 1—improved efficacy; 2—reduced risks of side effectsand 3—a mechanism of action which is difficult for the virus to overcomeby mutation.

[0022] Several attempts to inhibit particular viruses by variousantisense approaches have been made.

[0023] Zamecnik et al. have used ONs specifically targeted to thereverse transcriptase primer site and to splice donor/acceptor sites(Zamecnik, et al (1986) Proc. Natl. Acad. Sci. USA 83:4143-) (Goodchild& Zamecnik (1989) U.S. Pat. No. 4,806,463).

[0024] Crooke and coworkers. (Crooke et al. (1992) Antimicrob. AgentsChemother. 36:527-532) described an antisense against HSV-1.

[0025] Draper et al. (1993) (U.S. Pat. No. 5,248,670) have reportedantisense oligonucleotides having anti-HSV activity containing the Catsequence and hybridizing to the HSV-1 genes UL13, UL39 and UL40.

[0026] Kean et al. (Biochemistry (1995) 34:14617-14620) have testedantisense methylphosphonate oligomers as anti-HSV agents.

[0027] Peyman et al. (Biol Chem Hoppe Seyler (1995) Mar; 376:195-198)have reported testing specific antisense oligonucleotides directedagainst the IE110 and the UL30 mRNA of HSV-1 for their antiviralproperties.

[0028] Oligonucleotides or oligonucleotide analogs targeting CMV mRNAscoding for IE1, IE2 or DNA polymerase were reported by Anderson et al(1997) (U.S. Pat. No. 5,591,720)

[0029] Hanecak et al (1999) (U.S. Pat. No. 5,952,490) have describedmodified oligonucleotides having a conserved G quartet sequence and asufficient number of flanking nucleotides to significantly inhibit theactivity of a virus such as HSV-1.

[0030] Jairath et al (Antiviral Res. (1997) 33:201-213) have reportedantisense oligonucleotides against RSV.

[0031] Torrence et al (1999) (U.S. Pat. No. 5,998,602) have reportedcompounds comprising an antisense component complementary to a singlestranded portion of the RSV antigenomic strand (the mRNA strand), alinker and a oligonucleotide activator of RNase L.

[0032] Qi et al. (Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi(2000) 14:253-256) have reported testing antisense PS-ODNs in Coxsackievirus B3.

[0033] International publication WO9203051 (Roizman and Maxwell)describes methylphosphonate antisense oligomers which are complementaryto vital regions of HSV viral genome or mRNA transcripts thereof whichexhibit antiviral activity.

[0034] Guanosine/thymidine or guanosine-rich phophorothioateoligodeoxynucleotides (GT-PS-ODNs) have been reported to have antiviralactivity. The article stated that “several different PS-containingGT-rich ODNs (B106-140, I100-12, and G106-57) all 26 or 27 nt in length,were just as effective at reducing HIV-2 titers as GT-rich ODNsconsisting of 36 (B106-96, B106-97) or 45 nt (Table 4).” (Fennewald etal., Antiviral Res. (1995) 26:37-54).

[0035] In U.S. Pat. No. 6,184,369, anti-HIV, anti-HSV, and anti-CMVoligonucleotides containing a high percentage of guanosine bases aredescribed. In preferred embodiments, the oligonucleotide has a threedimensional structure and this structure is stabilized by guanosinetetrads. In a further embodiment, the oligonucleotide compositions ofthe invention have two or more runs of two contiguous deoxyguanosinesThe patent claims a G-rich ODN that includes at least two G residues inat least two positions.

[0036] Cohen et al. (U.S. Pat. Nos. 5,264,423 and 5,276,019) describedthe inhibition of replication of HIV, and more particularly to PS-ODNanalogs that can be used to prevent replication of foreign nucleic acidsin the presence of normal living cells. Cohen et al describe antiviralactivity of antisense PS-ODNs specific to a viral sequence. They alsodescribe testing polyA, polyT and polyC PS-ODN sequences of 14, 18, 21and 28-mers and indicate an antiviral effect of those PS-ODNs.

[0037] Matsukura et al. (Matsukura et al (1987) Proc Natl Acad Sci USA84:7706-7710) later published the result described in Cohen et al, USpatents above.

[0038] Gao et al (Gao et al (1989) J Biol Chem 264 :11521-11526),describe the inhibition of replication of HSV-2, by PS-ODNs by testingof polyA, polyT and polyC PS-ODN sequences in sizes of 7, 15, 21 and 28nucleotides.

[0039] Archambault, Stein and Cohen (Archambault et al (1994) Arch Virol139:97109) report that a PS-ODN polyC of 28 nucleotides is not effectiveagainst HSV-1.

[0040] Stein et al (Stein et al. (1989) AIDS Res Hum Retrovir5:639-646), published results concerning additional data on anti-HIVODNs, generally of 21-28 nucleotides in length.

[0041] Marshal et al. (Marshall et al. (1992) Proc. Natl. Acad. Sci. USA89:6265-6269) describe anti-HIV-1 effect of phosphorothioate andphosphorothioate polyC oligos of 4-28 nucleotides in length.

[0042] Stein & Cheng (Stein et al. (1993) Science 261 :1004-1012), in areview article, mention the antiviral activity of non specific ODNs of28 nucleotides, stating that “the anti-HIV properties of PS oligos aresignificantly influenced by non-sequence-specific effects, that is, theinhibitory effect is independent of the base sequence.”

[0043] In a review article Lebedeva & Stein (Lebedeva et al (2001) AnnulRev Pharmacol 41:403-419) report a variety of non-specific proteinbinding activity of PS-ODNs, including viral proteins. They state that“these molecules are highly biologically active, and it is oftenrelatively easy to mistake artifact for antisense”.

[0044] Rein et al. (U.S. Pat. No. 6,316,190) reported a GT rich ON decoylinked to a fusion partner and binding to the HIV nucleocapsid, whichcan be used as an antiviral compound. Similarly, Campbell et al.(Campbell et al (1999) J. Virol. 73 :2270-2279) reported PO-ODN with aTGTGT motif binding specifically to the nucleocapsid of HIV but with noreferences to an antiviral activity.

[0045] Feng at al. (Feng et al. (2002) J. Virol. 76 :11757-11762)described A(n) and TG(n) PO-ODNs binding to the recombinant HIVnucleocapsid but with no data nor references to an anti-HIV activity.

[0046] Antisense ODNs developed as anticancer agents, antiviral agents,or to treat others diseases are typically approximately 20 nucleotidesin length. In a review article (Stein, C A, (2001) J. Clin. Invest.108:641-644), it is affirmed that “the length of an antisenseoligonucleotide must be optimized: If the antisense oligonucleotide iseither too long or too short, an element of specificity is lost. At thepresent time, the optimal length for an antisense oligonucleotide seemsto be roughly 16-20 nucleotides”. Similarly, in another review article(Crooke, ST (2000) Methods Enzymol. 313:3-45) it is stated that“Compared to RNA and RNA duplex formation, a phosphorothioateoligodeoxynucleotide has a T_(m) approximately −2.2° lower per unit.This means that to be effective in vitro, phosphorothioateoligodeoxynucleotides must typically be 17- to −20-mer in length . . .”.

[0047] Caruthers and co-workers (Marshall et al. (1992) Proc. Natl.Acad. Sci. USA 89:6265-6269) reported anti-HIV activity ofphosphorodithioate ODNs (PS2-ODNs) for a 12mer polycytidine-PS2-ODN andfor a 14mer PS2-ODN. No other sizes were tested for anti-HIV activity.They also reported the inhibition of HIV reverse transcriptase (RT) for12, 14, 20 and 28mer polycytidine-PS2-ODNs. Later, (Marshal et al (1993)Science 259:1564-1570) reported results showing sequence specificinhibition of the HIV RT. The same group published data for PS2-ODNs inseveral patents. In U.S. Pat. Nos. 5,218,103 and 5,684,148, PS2-ODNstructure and synthesis is described. In U.S. Pat. Nos. 5,452,496,5,278,302, and 5,695,979 inhibition of HIV RT is described for PS2-ODNsnot longer than 15 bases. In U.S. Pat. Nos. 5,750,666 and 5,602,244,antisense activity of PS2-ODNs is described.

SUMMARY OF THE INVENTION

[0048] The present invention involves the discovery thatoligonucleotides (ONs), e.g., oligodeoxynucleotides (ODNs), can have abroadly applicable, non-sequence complementary antiviral activity. Thus,it is not necessary for the oligonucleotide to be complementary to anyviral sequence or to have a particular distribution of nucleotides inorder to have antiviral activity. Such an oligonucleotide can even beprepared as a randomer, such that there will be at most a few copies ofany particular sequence in a preparation, e.g., in a 15 micromolrandomer preparation 32 or more nucleotides in length.

[0049] In addition, the inventors discovered that different lengtholigonucleotides have varying antiviral effect, and further that thelength of antiviral oligonucleotide that produces maximal antiviraleffect is in the range of 40-120 nucleotides. In view of the presentdiscoveries concerning antiviral properties of oligonucleotides, thisinvention provides oligonucleotide antiviral agents that can haveactivity against numerous different viruses, and can even be selected asbroad-spectrum antiviral agents. Such antiviral agents are particularlyadvantageous in view of the limited antiviral therapeutic optionscurrently available.

[0050] Therefore, the ONs, e.g., ODNs, of the present invention areuseful in therapy for treating or preventing viral infections or fortreating or preventing tumors or cancers induced by viruses, such asoncoviruses (e.g., retroviruses, papillomaviruses, and herpesviruses),and in treating or preventing other diseases whose etiology isviral-based. Such treatments are applicable to many types of patientsand treatments, including, for example, the prophylaxis or treatment ofviral infections in immunosuppressed human and animal patients.

[0051] In a first aspect, the invention provides an antiviraloligonucleotide formulation that includes at least one antiviraloligonucleotide, e.g., at least 6 nucleotides in length, adapted for useas an antiviral agent, where the antiviral activity of theoligonucleotide occurs principally by a non-sequence complementary modeof action. Such a formulation can include a mix of differentoligonucleotides, e.g., at least 2, 3, 5, 10, 50, 100, or even more.

[0052] As used herein in connection with oligonucleotides or othermaterials, the term “antiviral” refers to an effect of the presence ofthe oligonucleotides or other material in inhibiting production of viralparticles, i.e., reducing the number of infectious viral particlesformed, in a system otherwise suitable for formation of infectious viralparticles for at least one virus. In certain embodiments of the presentinvention, the antiviral oligonucleotides will have antiviral activityagainst multiple different virus.

[0053] The term “antiviral oligonucleotide formulation” refers to apreparation that includes at least one antiviral oligonucleotide that isadapted for use as an antiviral agent. The formulation includes theoligonucleotide or oligonucleotides, and can contain other materialsthat do not interfere with use as an antiviral agent in vivo. Such othermaterials can include without restriction diluents, excipients, carriermaterials, and/or other antiviral materials.

[0054] As used herein, the term “pharmaceutical composition” refers toan antiviral oligonucleotide formulation that includes a physiologicallyor pharmaceutically acceptable carrier or excipient. Such compositionscan also include other components that do not make the compositionunsuitable for administration to a desired subject, e.g., a human.

[0055] In the context of the present invention, unless specificallylimited the term “oligonucleotide (ON)” means oligodeoxynucleotide (ODN)or oligodeoxyribonucleotide or oligoribonucleotide. Thus,“oligonucleotide” refers to an oligomer or polymer of ribonucleic acid(RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This termincludes oligonucleotides composed of naturally-occurring nucleobases,sugars and covalent internucleoside (backbone) linkages as well asoligonucleotides having non-naturally-occurring portions which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for nucleic acidtarget and increased stability in the presence of nucleases. Examples ofmodifications that can be used are described herein. Oligonucleotidesthat include backbone and/or other modifications can also be referred toas oligonucleosides.

[0056] As used in connection with an antiviral formulation,pharmaceutical composition, or other material, the phrase “adapted foruse as an antiviral agent” indicates that the material exhibits anantiviral effect and does not include any component or material thatmakes it unsuitable for use in inhibiting viral production in an in vivosystem, e.g., for administering to a subject such as a human subject.

[0057] As used herein in connection with antiviral action of a material,the phrase “non-sequence complementary mode of action” indicates thatthe mechanism by which the material exhibits an antiviral effect is notdue to hybridization of complementary nucleic acid sequences, e.g., anantisense effect. Conversely, a “sequence complementary mode of action”means that the antiviral effect of a material involves hybridization ofcomplementary nucleic acid sequences. Thus, indicating that theantiviral activity of a material is “not primarily due to a sequencecomplementary mode of action” means that the the activity of theoligonucleotide satisfies at least one of the 4 tests provided herein(see Example, 10) for determining whether the antiviral activity is “notprimarily due to a sequence complementary mode of action”. In particularembodiments, the oligonucleotide satisfies test 1, test 2, test 3, ortest 4; the oligonucleotide satisfies a combination of two of the tests,i.e., tests 1 & 2; tests 1 & 3; tests 1 & 4, tests 2 & 3, tests 2 & 4,or tests 3 & 4; the oligonucleotide satisfies a combination of 3 of thetests, i.e., tests 1, 2, and 3, tests 1, 2, and 4, tests 1, 3, and 4, ortests 2, 3, and 4; the oligonucleotide satisifies all of tests 1, 2, 3,and 4.

[0058] As used herein in connection with administration of an antiviralmaterial, the term “subject” refers to a living higher organism,including, for example, animals such as mammals, e.g., humans, non-humanprimates, bovines, porcines, ovines, equines, dogs, and cats; birds; andplants, e.g., fruit trees.

[0059] A related aspect concerns an antiviral oligonucleotide randomerformulation, where the antiviral activity of the randomer occursprincipally by a non-sequence complementary mode of action. Such arandomer formulation can, for example, include a mixture of randomers ofdifferent lengths, e.g., at least 2, 3, 5, 10, or more differentlengths.

[0060] As used herein in connection with oligonucleotide sequences, theterm “random” characterizes a sequence or an ON that is notcomplementary to a viral mRNA, and which is selected to not formhairpins and not to have palindromic sequences contained therein. Whenthe term “random” is used in the context of antiviral activity of anoligonucleotide toward a particular virus, it implies the absence ofcomplementarity to a viral mRNA of that particular virus. The absence ofcomplementarity may be broader, e.g., for a plurality of viruses, forviruses from a particular viral family, or for infectious human viruses.

[0061] In the present application, the term “randomer” is intended tomean a single stranded DNA having a wobble (N) at every position, suchas NNNNNNNNNN. Each base is synthesized as a wobble such that this ONactually exists as a population of different randomly generatedsequences of the same size.

[0062] In another aspect, the invention provides an oligonucleotidehaving antiviral activity against a target virus, where theoligonucleotide is at least 29 nucleotides in length (or in particularembodiments, at least 30, 32, 34, 36, 38, 40, 46, 50, 60, 70, 80, 90,100, 110, or 120 nucleotides in length) and the sequence of theoligonucleotide is not complementary to any portion of the genomesequence of the target virus.

[0063] In another aspect, the invention provides an oligonucleotideformulation, containing at least one oligonucleotide having antiviralactivity against a target virus, where the oligonucleotide is at least 6nucleotides in length (in particular embodiments, at least 29, 30, 32,34, 36, 38, 40, 46, 50, 60, 70, 80, 90, 100, 110, or 120 nucleotides inlength) and the sequence of oligonucleotide is less than 70%complementary to any portion of the genomic nucleic acid sequence of thetarget virus and does not consist essentially of polyA, polyC, polyG,polyT, Gquartet, or a TG-rich sequence. In particular embodiments, theoligonucleotide has less than 65%, 60%, 55%, 50%, 80% 90%, 95%, or 100%complementarity to any portion of the genomic nucleic acid sequence ofthe target virus.

[0064] As used in connection with the present oligos, the term “TG-rich”indicates that the sequence of the antiviral oligonucleotide consists ofat least 70 percent T and G nucleotides, or if so specified, at least80, 90, or 95% T and G, or even 100%.

[0065] Related aspects concern isolated, purified or enriched antiviraloligonucleotides as described herein, e.g., as described for antiviraloligonucleotide formulations, as well as other oligonucleotidepreparations, e.g., preparations suitable for in vitro use.

[0066] Antiviral oligonucleotides useful in the present invention can beof various lengths, e.g., at least 6, 10, 14, 15, 20, 25, 28, 29, 30,35, 38, 40, 46, 50, 60, 70, 80, 90, 100, 110, 120, 140, 160, or morenucleotides in length. Likewise, the oligonucleotide can be in a range,e.g., a range defined by taking any two of the preceding listed valuesas inclusive end points of the range, for example 10-20, 20-40, 30-50,40-60, 40-80, 60-120, and 80-120 nucleotides. In particular embodiments,a minimum length or length range is combined with any other of theoligonucleotide specifications listed herein for the present antiviraloligonucleotides.

[0067] The antiviral nucleotide can include various modifications, e.g.,stabilizing modifications, and thus can include at least onemodification in the phosphodiester linkage and/or on the sugar, and/oron the base. For example, the oligonucleotide can include one or morephosphorothioate linkages, phosphorodithioate linkages, and/ormethylphosphonate linkages; modifications at the 2′-position of thesugar, such as 2′-O-methyl modifications, 2′-amino modifications,2′-halo modifications such as 2′-fluoro; acyclic nucleotide analogs, andcan also include at least one phosphodiester linkage. Othermodifications are also known in the art and can be used. In oligos thatcontain 2′-O-methyl modifications, the oligo should not have 2′-O-methylmodifications throughout, as current results suggest that such oligos donot have suitable activity. In particular embodiments, theoligonucleotide has modified linkages throughout, e.g.,phosphorothioate; has a 3′- and/or 5′-cap; includes a terminal 3′-5′linkage; the oligonucleotide is or includes a concatemer consisting oftwo or more oligonucleotide sequences joined by a linker(s)

[0068] In particular embodiments, the oligonucleotide binds to one ormore viral proteins; the sequence of the oligonucleotide (or a portionthereof, e.g., at least ½) is derived from a viral genome; the activityof an oligonucleotide with a sequence derived from a viral genome is notsuperior to a randomer oligonucleotide or a random oligonucleotide ofthe same length; the oligonucleotide includes a portion complementary toa viral sequence and a portion not complementary to a viral sequence;the sequence of the oligonucleotide is derived from a viral packagingsequence or other viral sequence involved in an aptameric interaction;unless otherwise indicated, the sequence of the oligonucleotide includesA(x), C(x), G(x), T(x), AC(x), AG(x), AT(x), CG(x), CT(x), or GT(x),where x is 2, 3, 4, 5, 6, . . . 60 . . . 120 (in particular embodimentsthe oligonucleotide is at least 29, 30, 32, 34, 36, 38, 40, 46, 50, 60,70, 80, 90, 100, 110, or 120 nucleotides in length or the length of thespecified repeat sequence is at least a length just specified); theoligonucleotide is single stranded (RNA or DNA); the oligonucleotide isdouble stranded (RNA or DNA); the oligonucleotide includes at least oneGquartet or CpG portion; the oligonucleotide includes a portioncomplementary to a viral mRNA and is at least 29, 37, or 38 nucleotidesin length (or other length as specified above); the oligonucleotideincludes at least one non-Watson-Crick oligonucleotide and/or at leastone nucleotide that participates in non-Watson-Crick binding withanother nucleotide; the oligonucleotide is a random oligonucleotide, theoligonucleotide is a randomer or includes a randomer portion, e.g., arandomer portion that has a length as specified above foroligonucleotide length; the oligonucleotide is linked or conjugated atone or more nucleotide residues to a molecule that modifies thecharacteristics of the oligonucleotide, e.g. to provide higher stability(such as stability in serum or stability in a particular solution),lower serum interaction, higher cellular uptake, higher viral proteininteraction, improved ability to be formulated for delivery, adetectable signal, improved pharmacokinetic properties, specific tissuedistribution, and/or lower toxicity.

[0069] Oligonucleotides can also be used in combinations, e.g., as amixture. Such combinations or mixtures can include, for example, atleast 2, 4, 10, 100, 1000, 10000, 100,000, 1,000,000, or more differentoligonucleotides. Such combinations or mixtures can, for example, bedifferent sequences and/or different lengths and/or differentmodifications and/or different linked or conjugated molecules. Inparticular embodiments of such combinations or mixtures, a plurality ofoligonucleotides have a minimum length or are in a length range asspecified above for oligonucleotides. In particular embodiments of suchcombinations or mixtures, at least one, a plurality, or each of theoligonucleotides can have any of the other properties specified hereinfor individual antiviral oligonucleoties (which can also be in anyconsistent combination).

[0070] The phrase “derived from a viral genome” indicates that aparticular sequence has a nucleotide base sequence that has at least 70%identity to a viral genomic nucleotide sequence or its complement (e.g.,is the same as or complementary to such viral genomic sequence), or is acorresponding RNA sequence. In particular embodiments of the presentinvention, the term indicates that the sequence is at least 70%identical to a viral genomic sequence of the particular virus againstwhich the oligonucleotide is directed, or to its complementary sequence.In particular embodiments, the identity is at least 80, 90, 95, 98, 99,or 100%.

[0071] The invention also provides an antiviral pharmaceuticalcomposition that includes a therapeutically effective amount of apharmacologically acceptable, antiviral oligonucleotide at least 6nucleotides in length (or other length as listed herein), where theantiviral activity of the oligonucleotide occurs principally by anon-sequence complementary mode of action, and a pharmaceuticallyacceptable carrier. In particular embodiments, the oligonucleotide or acombination or mixture of oligonucleotides is as specified above forindividual oligonucleotides or combinations or mixtures ofoligonucleotides. In particular embodiments, the pharmaceuticalcompositions are approved for administration to a human, or a non-humananimal such as a non-human primate.

[0072] In particular embodiments, the pharmaceutical composition isadapted for the treatment, control, or prevention of a disease with aviral etiology; adapted for treatment, control, or prevention of a priondisease; is adapted for delivery by intraocular administration, oralingestion, enteric administration, inhalation, cutaneous, subcutaneous,intramuscular, intraperitoneal, intrathecal, intratracheal, orintravenous injection, or topical administration. In particularembodiments, the composition includes a delivery system, e.g., targetedto specific cells or tissues; a liposomal formulation, another antiviraldrug, e.g., a non-nucleotide antiviral polymer, an antisense molecule,an siRNA, or a small molecule drug.

[0073] In particular embodiments, the antiviral oligonucleotide,oligonucleotide preparation, oligonucleotide formulation, or antiviralpharmaceutical composition has an IC50 for a target virus (e.g., any ofparticular viruses or viruses in a groups of viruses as indicatedherein) of 0.50, 0.20, 0.10, 0.09, 0.08, 0.07, 0.75, 0.06, 0.05, 0.045,0.04, 0.035, 0.03, 0.025, 0.02, 0.015, or 0.01 μM or less.

[0074] In particular embodiments of formulations, pharmaceuticalcompositions, and methods for prophylaxis or treatment, the compositionor formulation is adapted for treatment, control, or prevention of adisease with viral etiology; is adapted for the treatment, control orprevention of a prion disease; is adapted for delivery by a modeselected from the group consisting of intraocular, oral ingestion,enterally, inhalation, or cutaneous, subcutaneous, intramuscular, orintravenous injection delivery; further comprises a delivery system,which can include or be associated with a molecule increasing affinitywith specific cells; further comprises at least one other antiviral drugin combination; and/or further comprises an antiviral polymer incombination.

[0075] As used herein in connection with antiviral oligonucleotides andformulations, and the like, in reference to a particular virus or groupof viruses the term “targeted” indicates that the oligonucleotide isselected to inhibit that virus or group of viruses. As used inconnection with a particular tissue or cell type, the term indicatesthat the oligonucleotide, formulation, or delivery system is selectedsuch that the oligonucleotide is preferentially present and/orpreferentially exhibits an antiviral effect in or proximal to theparticular tissue or cell type.

[0076] As used herein, the term “delivery system” refers to a componentor components that, when combined with an oligonucleotide as describedherein, increases the amount of the oligonucleotide that contacts theintended location in vivo, and/or extends the duration of its presenceat the target, e.g., by at least 20, 50, or 100%, or even more ascompared to the amount and/or duration in the absence of the deliverysystem, and/or prevents or reduces interactions that cause side effects.

[0077] As used herein in connection with antiviral agents and otherdrugs or test compounds, the term “small molecule” means that themolecular weight of the molecule is 1500 daltons or less. In some cases,the molecular weight is 1000, 800, 600, 500, or 400 daltons or less.

[0078] In another aspect, the invention provides a kit that includes atleast one antiviral oligonucleotide or oligonucleotide formulation in alabeled package, where the antiviral activity of the oligonucleotideoccurs principally by a non-sequence complementary mode of action andthe label on the package indicates that the antiviral oligonucleotidecan be used against at least one virus.

[0079] In particular embodiments the kit includes a pharmaceuticalcomposition that includes at least one antiviral oligonucletide asdescribed herein; the antiviral oligonucleotide is adapted for in vivouse in an animal and/or the label indicates that the oligonucleotide orcomposition is acceptable and/or approved for use in an animal; theanimal is a mammal, such as human, or a non-human mammal such as bovine,porcine, a ruminant, ovine, or equine; the animal is a non-human animal;the kit is approved by a regulatory agency such as the U.S. Food andDrug Administration or equivalent agency for use in an animal, e.g., ahuman.

[0080] In another aspect, the invention provides a method for selectingan antiviral oligonucleotide, e.g, a non-sequence complementaryantiviral oligonucleotide, for use as an antiviral agent. The methodinvolves synthesizing a plurality of different random oligonucleotides,testing the oligonucleotides for activity in inhibiting the ability of avirus to produce infectious virions, and selecting an oligonucleotidehaving a pharmaceutically acceptable level of activity for use as anantiviral agent.

[0081] In particular embodiments, the different random oligonucleotidescomprises randomers of different lengths; the random oligonucleotidescan have different sequences or can have sequence in common, such as thesequence of the shortest oligos of the plurality; and/or the differentrandom oligonucleotides comprise a plurality of oligonucleotidescomprising a randomer segment at least 5 nucleotides in length or thedifferent random oligonucleotides include a plurality of randomers ofdifferent lengths. Other oligonucleotides, e.g., as described herein forantiviral oligonucleotides, can be tested in a particular system.

[0082] In yet another aspect, the invention provides a method for theprophylaxis or treatment of a viral infection in a subject byadministering to a subject in need of such treatment a therapeuticallyeffective amount of at least one pharmacologically acceptableoligonucleotide as described herein, e.g., a non-sequence complementaryoligonucleotide at least 6 nucleotides in length, or an antiviralpharmaceutical composition or formulation containing sucholigonucleotide. In particular embodiments, the virus can be any ofthose listed herein as suitable for inhibition using the presentinvention; the infection is related to a disease or condition indicatedherein as related to a viral infection; the subject is a type of subjectas indicated herein, e.g., human, non-human animal, non-human mammal,plant, and the like; the treatment is for a viral disease or diseasewith a viral etiology, e.g., a disease as indicated in the Backgroundherein.

[0083] In particular embodiments, an antiviral oligonucleotide (oroligonucleotide formulation or pharmaceutical composition) as describedherein is administered; administration is a method as described herein;a delivery system or method as described herein is used; the viralinfection is of a DNA virus or an RNA virus; the virus is aparvoviridae, papovaviridae, adenoviridae, herpesviridae, poxyiridae,hepadnaviridae, or papillomaviridae; the virus is a arenaviridae,bunyaviridae, calciviridae, coronaviridae, filoviridae, flaviridae,orthomyxoviridae, paramyxoviridae, picornaviridae, reoviridae,rhabdoviridae, retroviridae, or togaviridae; the herpesviridae virus isEBV, HSV-1, HSV-2, CMV, VZV, HHV-6, HHV-7, or HHV-8; the virus is HIV-1or HIV-2; the virus is RSV; the virus is an influenza virus, e.g.,influenza A; the virus is HBV; the virus is smallpox virus or vacciniavirus; the virus is a coronavirus; the virus is SARS virus; the virus isWest Nile Virus; the virus is a hantavirus; the virus is a parainfluenzavirus; the virus is coxsackievirus; the virus is rhinovirus; the virusis yellow fever virus; the virus is dengue virus; the virus is hepatitisC virus; the virus is Ebola virus; the virus is Marburg virus.

[0084] Similarly, in a related aspect, the invention provides a methodfor the prophylactic treatment of cancer caused by oncoviruses in ahuman or animal by administering to a human or animal in need of suchtreatment, a pharmacologically acceptable, therapeutically effectiveamount of at least one random oligonucleotide of at least 6 nucleotidesin length (or another length as described herein), or a formulation orpharmaceutical composition containing such oligonucleotide.

[0085] In particular embodiments, the oligonucleotide(s) is as describedherein for the present invention, e.g., having a length as describedherein; a method of administration as described herein is used; adelivery system as described herein is used.

[0086] The term “therapeutically effective amount” refers to an amountthat is sufficient to effect a therapeutically or prophylacticallysignificant reduction in production of infectious virus particles whenadministered to a typical subject of the intended type. In aspectsinvolving administration of an antiviral oligonucleotide to a subject,typically the oligonucleotide, formulation, or composition should beadministered in a therapeutically effective amount.

[0087] In another aspect, the discovery that non-sequence complementaryinteractions produce effective antiviral activity provides a method ofscreening to identify a compound that alters binding of anoligonucleotide to a viral component, such as one or more viral proteins(e.g., extracted or purified from a viral culture of infected hostorganisms, or produced by recombinant methods). For example, the methodcan involve determining whether a test compound reduces the binding ofoligonucleotide to one or more viral components.

[0088] As used herein, the term “screening” refers to assaying aplurality of compounds to determine if they possess a desired property.The plurality of compounds can, for example, be at least 10, 100, 1000,10,000 or more test compounds.

[0089] In particular embodiments, any of a variety of assay formats anddetection methods can be used to identify such alteration in binding,e.g., by contacting the oligonucleotide with the viral component(s) inthe presence and absence of a compound(s) to be screened (e.g., inseparate reactions) and determining whether a difference occurs inbinding of the oligo the viral component(s) in the presence of thecompound compared to the absence of the compound. The presence of such adifference is indicative that the compound alters the binding of therandom oligonucleotide to the viral component. Alternatively, acompetitive displacement can be used, such that oligonucleotide is boundto the viral component and displacement by added test compound isdetermined, or conversely test compound is bound and displacement byadded oligonucleotide is determined.

[0090] In particular embodiments, the oligonucleotide is as describedherein for antiviral oligonucleotides; the oligonucleotide is at least6, 8, 10, 15, 20, 25, 29, 30, 32, 34, 36, 38, 40, 46, 50, 60, 70, 80,90, 100, 110, or 120 nucleotides in length or at least another lengthspecified herein for the antiviral oligonucleotides, or is in a rangedefined by taking any two of the preceding values as inclusive endpointsof the range; the test compound(s) is a small molecule; the testcompound has a molecular weight of less than 400, 500, 600, 800, 1000,1500, 2000, 2500, or 3000 daltons, or is in a range defined by takingany two of the preceding values as inclusive endpoints of the range; theviral extract or component is from a virus as listed herein; at least100, 1000, 10,000, 20,000, 50,000, or 100,000 compounds are screened;the oligonucleotide has an IC50 of equal to or less than 0.500, 0.200,0.100, 0.075, 0.05, 0.045, 0.04, 0.035, 0.03, 0.025, 0.02, 0.015, or0.01 μM.

[0091] As used herein, the term “viral component” refers to a productencoded by a virus or produced by infected host cells as a consequenceof the viral infection. Such components can include proteins as well asother biomolecules. Such viral components, can, for example, be obtainedfrom viral cultures, infected host organisms, e.g., animals and plants,or can be produced from viral sequences in recombinant systems(prokaryotes and eukaryotes), as well synthetic proteins having aminoacid sequences corresponding to viral encoded proteins. The term “viralculture extract” refers to an extract from cells infected by a virusthat will include virus-specific products. Similarly, a “viral protein”refers to a virus-specific protein, usually encoded by a virus, but canalso be encoded at least in part by host sequences as a consequence ofthe viral infection.

[0092] In a related aspect, the invention provides an antiviral compoundidentified by the preceding method, e.g., a novel antiviral compound.

[0093] In a further aspect, the invention provides a method forpurifying oligonucleotides binding to at least one viral component froma pool of oligonucleotides by contacting the pool with at least oneviral component, e.g., bound to a stationary phase medium, andcollecting oligonucleotides that bind to the viral component(s).Generally, the collecting involves displacing the oligonucleotides fromthe viral component(s). The method can also involve sequencing and/ortesting antiviral activity of collected oligonucleotides (i.e.,oligonucleotides that bound to viral protein).

[0094] In particular embodiments, the bound oligonucleotides of the poolare displaced from the stationary phase medium by any appropriatemethod, e.g., using an ionic displacer, and displaced oligonucleotidesare collected. Typically for the various methods of displacement, thedisplacement can be performed in increasing stringent manner (e.g., withan increasing concentration of displacing agent, such as a saltconcentration, so that there is a stepped or continuous gradient), suchthat oligonucleotides are displaced generally in order of increasedbinding affinity. In many cases, a low stringency wash will be performedto remove weakly bound oligonucleotides, and one or more fractions willbe collected containing displaced, tighter binding oligonucleotides. Insome cases, it will be desired to select fractions that contain verytightly binding oligonucleotides (e.g., oligonucleotides in fractionsresulting from displacement by the more stringent displacementconditions) for further use.

[0095] Similarly, the invention provides a method for enrichingoligonucleotides from a pool of oligonucleotides binding to at least oneviral component, by contacting the pool with one or more viral proteins,and amplifying oligonucleotides bound to the viral proteins to providean enriched oligonucleotide pool. The contacting and amplifying can beperformed in multiple rounds, e.g., at least 1, 2, 3, 4, 5, 10, or moreadditional times using the enriched oligonucleotide pool from thepreceding round as the pool of oligonucleotides for the next round. Themethod can also involve sequencing and testing antiviral activity ofoligonucleotides in the enriched oligonucleotide pool following one ormore rounds of contacting and amplifying.

[0096] The method can involve displacing oligonucleotides from the viralcomponent (e.g., viral protein bound to a solid phase medium) with anyof a variety of techniques, such as those described above, e.g., using adisplacement agent. As indicated above, it can be advantageous to selectthe tighter binding oligonucleotides for further use, e.g., in furtherrounds of binding and amplifying. The method can further involveselecting one or more enriched oligonucleotides, e.g., high affinityoligonucleotides, for further use. In particular embodiments, theselection can include eliminating oligonucleotides that have sequencescomplementary to host genomic sequences (e.g., human) for a particularvirus of interest. Such elimination can involve comparing theoligonucleotide sequence(s) with sequences from the particular host in asequence database(s), e.g., using a sequence alignment program (e.g., aBLAST search), and eliminating those oligonucleotides that havesequences identical or with a particular level of identity to a hostsequence. Eliminating such host complementary sequences and/or selectingone or more oligonucleotides that are not complementary to hostsequences can also be done for the other aspects of the presentinvention.

[0097] In the preceding methods for identifying, purifying, or enrichingoligonucleotides, the oligonucleotides can be of types as describedherein. The above methods are advantageous for identifying, purifying orenriching high affinity oligonucleotides, e.g., from an oligonucleotiderandomer preparation.

[0098] In a related aspect, the invention concerns an antiviraloligonucleotide preparation that includes one or more oligonucleotidesidentified using a method of any of the preceding methods foridentifying, obtaining, or purifying antiviral oligonucleotides from aninitial oligonucleotide pool, where the oligonucleotides in theoligonucleotide preparation exhibit higher mean binding affinity withone or more viral proteins than the mean binding affinity ofoligonucletides in the initial oligonucleotide pool.

[0099] In particular embodiments, the mean binding affinity of theoligonucleotides is at least two-fold, 3-fold, 5-fold, 10-fold, 20-fold,50-fold, or 100-fold greater than the mean binding affinity ofoligonucleotides in the initial oligonucleotide pool, or even more; themedian of binding affinity is at least two-fold, 3-fold, 5-fold,10-fold, 20-fold, 50-fold, or 100-fold greater relative to the median ofthe binding affinity of the initial oligo pool, where median refers tothe middle value.

[0100] In yet another aspect, the invention provides an antiviralpolymer mix that includes at least one antiviral oligonucleotide and atleast one non-nucleotide antiviral polymer. In particular embodiments,the oligonucleotide is as described herein for antiviraloligonucleotides and/or the antiviral polymer is as described herein orotherwise known in the art or subsequently identified.

[0101] In yet another aspect, the invention provides an oligonucleotiderandomer, where the randomer is at least 6 nucleotides in length. Inparticular embodiments the randomer has a length as specified above forantiviral oligonucleotides; the randomer includes at least onephosphorothioate linkage, the randomer includes at least onephosphorodithioate linkage or other modification as listed herein; therandomer oligonucleotides include at least one non-randomer segment(such as a segment complementary to a selected virus nucleic acidsequence), which can have a length as specified above foroligonucleotides; the randomer is in a preparation or pool ofpreparations containing at least 5, 10, 15, 20, 50, 100, 200, 500, or700 micromol, 1, 5, 7, 10, 20, 50, 100, 200, 500, or 700 mmol, or 1 moleof randomer, or a range defined by taking any two different values fromthe preceding as inclusive end points, or is synthesized at one of thelisted scales or scale ranges.

[0102] Likewise, the invention provides a method for preparing antiviralrandomers, by synthesizing at least one randomer, e.g., a randomer asdescribed above.

[0103] As indicated above, for any aspect involving a viral infection orrisk of viral infection or targeting to a particular virus, inparticular embodiments the virus is as listed above.

[0104] The expression “human and animal viruses” is intended to include,without limitation, DNA and RNA viruses in general. DNA viruses include,for example, parvoviridae, papovaviridae, adenoviridae, herpesviridae,poxyiridae, hepadnaviridae, and papillomaviridae. RNA viruses include,for example, arenaviridae, bunyaviridae, calciviridae, coronaviridae,filoviridae, flaviridae, orthomyxoviridae, paramyxoviridae,picornaviridae, reoviridae, rhabdoviridae, retroviridae, or togaviridae.

[0105] In connection with modifying characteristics of anoligonucleotide by linking or conjugating with another molecule ormoiety, the modifications in the characteristics are evaluated relativeto the same oligonucleotide without the linked or conjugated molecule ormoiety.

[0106] Additional embodiments will be apparent from the DetailedDescription and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0107]FIG. 1. Plaque reduction assay conducted in VERO cells using HSV-1(strain KOS). Infected cells are treated with increasing concentrationsof REP 1001 (a), REP 2001 (b) or REP 3007 (c). IC₅₀ values calculatedfrom linear regressions are reported in each graph.

[0108]FIG. 2. Relationship between PS-ODN size and IC₅₀ against HSV-1.IC₅₀ values from FIG. 1 are plotted against the specific size of eachPS-ODN tested in FIG. 1.

[0109]FIG. 3. Plaque reduction assay conducted in VERO cells using HSV-1(strain KOS). Infected cells are treated with increasing concentrationsof REP 2001 (a), REP 2002 (b) or REP 3003 (c), REP 2004 (d), REP 2005(e), REP 2006 (f) and Acyclovir (g). IC₅₀ values calculated from linearregressions are reported in each graph.

[0110]FIG. 4. Relationship between PS-ODN size and IC₅₀ against HSV-1.IC₅₀ values from FIG. 3 are plotted against the specific size of eachPS-ODN tested in FIG. 3 which showed anti-HSV-1 activity. The IC₅₀ forAcyclovir is indicated for reference to a clinical correlate.

[0111]FIG. 5. Plaque reduction assay conducted in VERO cells using HSV-1(strain KOS). A broad range of PS-ODN randomer sizes were tested inincreasing concentrations; REP 2003 (a), REP 2009 (b), REP 2010 (c), REP2011 (d), REP 2012 (e), REP 2004 (f), REP 2006 (g), REP 2007 (h) and REP2008 (i). IC₅₀ values calculated from linear regressions are reported ineach graph.

[0112]FIG. 6. UV backshadowing of PS-ODN randomers tested in FIG. 5separated by acrylamide gel electrophoresis.

[0113]FIG. 7. Relationship between PS-ODN randomer size and IC₅₀ againstHSV-1. IC₅₀ values from FIG. 5 are plotted against the specific size ofeach PS-ODN tested in FIG. 5 which showed anti-HSV-1 activity.

[0114]FIG. 8. Plaque reduction assay conducted in VERO cells using HSV-1(strain KOS). Umodified ODNs, PS-ODNs with a random sequence and PS-ODNstargeting the start codon of HSV-1 IE110 were tested in increasingconcentrations. REP 2013 (a), REP 2014 (b), REP 2015 (c), REP 2016 (d),REP 2017 (e), REP 2018 (f), REP 2019 (g), REP 2020 (h) and REP 2021 (i).IC₅₀ values calculated from linear regressions are reported in eachgraph.

[0115]FIG. 9. UV backshadowing of PS-ODN randomers tested in FIG. 8separated by acrylamide gel electrophoresis.

[0116]FIG. 10. Relationship between PS-ODN randomer, PS-ODN randomsequence, PS-ODN HSV-1 IE110 sequence and IC₅₀ against HSV-1. IC₅₀values from FIG. 8 are plotted against the specific size of each PS-ODNtested in FIG. 8 which showed anti-HSV-1 activity. Additional IC₅₀values from FIG. 5 are included for comparison against PS-ODN randomers.

[0117]FIG. 11. Plaque reduction assay conducted in VERO cells usingHSV-1 (strain KOS). A PS-ODN having 2-0 methyl modifications to the 4ribose sugars at each end of the oligo (REP 2024, [a]); a ODN havingmethylphosphonate modifications to the 4 ester linkages at each end ofthe oligo (REP 2026 [b]); and RNA PS-ODNs 20 bases (REP2059 [c]) and 30bases (REP2060 [d]) in length were tested in increasing concentrations.IC₅₀ values calculated from linear regressions are reported in eachgraph.

[0118]FIG. 12. Plaque reduction assay conducted in human fibroblastcells using HSV-2 (strain MS2). Infected cells are treated withincreasing concentrations of REP 1001 (a), REP 2001 (b) or REP 3007 (c).IC₅₀ values calculated from linear regressions are reported in eachgraph.

[0119]FIG. 13. Relationship between PS-ODN size and IC₅₀ against HSV-2.IC₅₀ values from FIG. 12 are plotted against the specific size of eachPS-ODN tested in FIG. 12.

[0120]FIG. 14. Plaque reduction assay conducted in VERO cells usingHSV-2 (strain MS2). Infected cells are treated with increasingconcentrations of REP 2001 (a), REP 2002 (b) or REP 2003 (c), REP 2004(d), REP 2005 (e), REP 2006 (f) and acyclovir (g). IC₅₀ valuescalculated from linear regressions are reported in each graph.

[0121]FIG. 15. Relationship between PS-ODN size and IC₅₀ against HSV-2.IC₅₀ values from FIG. 14 are plotted against the specific size of eachPS-ODN tested in FIG. 14 which showed anti-HSV-2 activity. The IC₅₀ foracyclovir is provided for reference to a clinical correlate.

[0122]FIG. 16. Plaque reduction assay conducted in VERO cells using CMV(strain AD169). Infected cells are treated with increasingconcentrations of REP 2004 (a) or REP 2006 (b). IC₅₀ values calculatedfrom linear regressions are reported in each graph. The relationshipbetween PS-ODN size and IC₅₀ against CMV is plotted in (c). IC₅₀ valuesfrom figure (a) and (b) are plotted against the specific size of eachPS-ODN tested.

[0123]FIG. 17. Plaque reduction assay conducted in VERO cells using CMV(strain AD169). Three clinical CMV therapies were tested: Gancyclovir(a), Foscarnet (b) and Cidofovir (c). A broad range of PS-ODN randomersizes were also tested in increasing concentrations; REP 2003 (d), REP2004 (e), REP 2006 (f) and REP 2007 (g). Finally, REP 2036 (Vitravene)was tested as synthesized in house (h) and as commercially available(i). IC₅₀ values calculated from linear regressions are reported in eachgraph.

[0124]FIG. 18. Relationship between PS-ODN size and IC₅₀ against CMV.IC₅₀ values from FIG. 17 are plotted against the specific size of eachPS-ODN tested in FIG. 17 which showed anti-CMV activity.

[0125]FIG. 19. CPE assay conducted in MT4 cells using HIV-1 (strainNL4-3). Infected cells are treated with increasing concentrations of REP2004 (a) or REP 2006 (b). IC₅₀ values calculated from linear regressionsare reported in each graph. Cytotoxicity profiles in uninfected MT4cells are presented for REP 2004 (c) and REP 2006 (d).

[0126]FIG. 20. Relationship between PS-ODN size and IC₅₀ against HIV-1.IC₅₀ values from FIG. 1 are plotted against the specific size of eachPS-ODN tested in FIG. 1.

[0127]FIG. 21. Replication assay conducted in 293A cells usingrecombinant wild type HIV-1 NL4-3 (strain CNDO). Infected cells aretreated with increasing concentrations of Amprenavir (a), Indinavir (b),Lopinavir (c), Saquinavir (d), REP 2003 (e), REP 2004 (f), REP 2006 (g)and REP 2007 (h). Both curves (black and dotted lines) represent doseresponse curves against strain CNDO.

[0128]FIG. 22. (a) IC₅₀ values from FIG. 21 and (b), relationshipbetween PS-ODN size and IC₅₀ against recombinant HIV-1. IC₅₀ values from(a) are plotted against the specific size of each PS-ODN tested in FIG.21.

[0129]FIG. 23. Replication assay conducted in 293A cells usingrecombinant multi drug resistant HIV-1 (strain MDRC4). Infected cellsare treated with increasing concentrations of Amprenavir (a), Indinavir(b), Lopinavir (c), Saquinavir (d), REP 2003 (e), REP 2004 (f), REP 2006(g) and REP 2007 (h). Dose response curves for CNDO (wild type) areindicated in dotted lines and for MDRC4 (drug resistant) are indicatedin solid lines.

[0130]FIG. 24. (a) IC50 values from FIGS. 21 and 23 showing foldincreases in IC50 values between wild type (CNDO) and drug resistant(MDRC4) strains of recombinant HIV-1 and (b), plot of fold increasescalculated in (a).

[0131]FIG. 25. CPE assay conducted in Hep2 cells using RSV (strain A2).Infected cells are treated with increasing concentrations of REP 2004(a), REP 2006 (b), REP 2007 (c) or Ribavirin (d). IC₅₀ values calculatedfrom linear regressions are reported in each graph. Cytotoxicityprofiles in uninfected Hep2 cells are presented for REP 2004 (e), REP2006 (f), REP 2007 (g) or Ribavirin (h).

[0132]FIG. 26. Relationship between PS-ODN size and IC₅₀ against RSV.IC₅₀ values from FIG. 25 are plotted against the specific size of eachPS-ODN tested in FIG. 25 which showed anti-RSV activity.

[0133]FIG. 27. CPE assay conducted in LLC-MK2 cells using CoxsackievirusB2 (strain Ohio-1). Infected cells are treated with increasingconcentrations of REP 2006 (a). The cytotoxicity profile for REP 2006 isshown in (b).

[0134]FIG. 28. A) FP interaction assay showing the ability of PS-ODNrandomers (REP 2003, 2004, 2006 and 2007) to compete the interaction ofa 20 base PS-ODN randomer bait from FBS. Larger randomers compete moreefficiently. B) and C) Serum protection and improved delivery of REP2006in 293 A cells with DOTAP and Cytofectin. D) and E) Serum protection ofREP 2006 encapsulated with DOTAP or cytofectin measured by FP.

[0135]FIG. 29. Determination of viral lysate binding to baits ofdifferent sizes by fluorescence polarization. REP 2032-FL, REP 2003-FLand REP 2004-FL were tested for lysate binding in lysates from HSV-1(a), HIV-1 (b) or RSV (c).

[0136]FIG. 30. Determination of affinity of PS-ODN randomers for virallysates by fluorescence polarization. Using REP 2004-FL as the bait,complex formation with HSV-1 lysate (a), HIV-1 lysate (b) or RSV lysate(c) was challenged with increasing concentrations of REP 2003, REP 2004,REP 2006 or REP 2007.

[0137]FIG. 31. REP 2004-FL can bind to HIV-1 p24gag and HIV-1 gp41. Theability of REP 2004-FL to interact with increasing amounts of these twopurified proteins is tested by fluorescence polarization.

[0138]FIG. 32. Effect of bait size on p24 and gp41 binding. Baits ofincreasing sizes are tested for their ability to bind to p24gag and gp41by fluorescence polarization.

[0139]FIG. 33. The ability of double stranded PS-ODNs to bind to virallysates is tested by fluorescence polarization. Single stranded (ss) ordouble stranded (ds) phosphorothioated REP 2017 (fluorescently labeled)was prepared as well as its non-thioated analog (2017U). These baitswere tested for binding to HSV-1 and HIV-1 viral lysates.

[0140]FIG. 34. The delivery of fluorescently tagged PS-ODNs into cellswas measured by incubating 293A cells in the presence of 250 nM REP2004-FL for 4 h. Following the incubation, cells were lysed and therelative fluorescence released from the cells upon lysis was measured byfluorometry.

[0141]FIG. 35. The ability of 20-mer PS-ODNs of different sequencecompositions to bind to viral lysates is measured by fluorescencepolarization. PS-ODNs 3′ labeled with FITC are incubated in the presenceof lug of HSV-1 (a), HIV-1 (b) or RSV (c) lysates. The binding profilesfor these PS-ODNs is similar in all three viral lysates (see FIG. 35).

[0142]FIG. 36. Indirect determination of viral load in infectedsupernatants from vaccinia infected VERO cells by measuring the CPEinduced by these supernatants in naive cells. REP 2004, 2006 and 2007were tested at 10 uM while Cidofovir was tested at 50 uM).

[0143]FIG. 37. (A) IC50 values generated from a plaque reduction assayconducted in VERO cells using HSV-1 (strain KOS). Infected cells aretreated with increasing concentrations of REP 2006 (N40), REP 2028(G40), REP 2029 (A40), REP 2030 (T40), and REP 2031 (C40) to generateIC50 values. (B) HSV-1 PRA generated IC50 values of the following: N40(REP 2006), AC20 (REP 2055, TC20 (REP 2056), or AG20 (REP 2057).

[0144] Selected Abbr Viations

[0145] ON: Oligonucleotide

[0146] ODN: Oligodeoxynucleotide

[0147] PS: Phosphorothioate

[0148] PRA: Plaque reduction assay

[0149] PFU: Plaque forming unit

[0150] INF A: Influenza A virus

[0151] HIV: Human immunodeficiency virus (includes both HIV-1 and HIV-2if not specified)

[0152] HSV: Herpes simplex virus (includes both HSV-2 and HSV-3 if notspecified)

[0153] RSV: Respiratory syncytial virus

[0154] COX: Coxsackievirus

[0155] DHBV: Duck hepatitis B virus

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0156] The present invention is concerned with the identification anduse of antiviral oligonucleotides that act by a non-sequencecomplementary mechanism, and includes the discovery that for manyviruses, the antiviral activity is greater for larger oligonucleotides,and are typically optimal for oligonucleotides that are 40 nucleotidesor more in length.

[0157] As described in the Background, a number of antisenseoligonucleotides (ONs) have been tested for antiviral activity. However,such antisense ONs are sequence-specific, and typically are about 16-20nucleotides in length.

[0158] As demonstrated by the results in Examples 1 and 2, the antiviraleffect of random PS-ODNs is not sequence specific. Considering thevolumes and concentrations of PS-ODNs used in those tests, it is almosttheoretically impossible that a particular random sequence is present atmore than 1 copy in the mixture. This means than there can be noantisense effect in these PS-ODNs randomers. In the latter example,should the antiviral effect be caused by the sequence-specificity of thePS-ODNs, such effect would thus have to be caused by only one molecule,a result that does not appear possible. For example, for an ON randomer40 bases in length, any particular sequence in the population wouldtheoretically represent only ¼⁴⁰ or 8.27×10⁻²⁵ of the total fraction.Given that 1 mole=6.022×10²³ molecules, and the fact that our largestsynthesis is currently done at the 15 micromole scale, all possiblesequences will not be present and also, each sequence is present mostprobably as only one copy. Of course, one skilled in the art applyingthe teaching of the present invention could also use sequence specificONs, but utilize the non-sequence complementary activity discovered inthe present invention. Accordingly, the present invention is not to berestricted to non-sequence complementary ONs, but disclaims what hasbeen disclosed in the prior art regarding sequence-specific antisenseONs for treating viral infections.

[0159] For applicable viruses (including, for example, those for whichdata is described herein), as the size of the randomer increases, sodoes its antiviral potency. It should be pointed out that due tolimitations in current phosphoramidite-based DNA synthesis, the largerPS-ODNs (e.g., 80- and 120-mers) have a significant contamination offragments smaller than the desired size. The weaker effects (on a perbase basis) seen with larger oligos (80 and 120 bp) may reflect thelower concentration of full-length randomers in these populations andmay also reflect a decreased uptake into the cell. It may be possible toachieve much larger increases in antiviral activity if larger randomers(>40 bases) of reasonable purity (75% full length) were synthesized orpurified, and/or if the cellular uptake of any of these ODNs isfacilitated by a delivery system.

[0160] In the present invention, randomers (or other oligonucleotides)may block viral replication by several mechanisms, including but notlimited to the following: 1. preventing the adsorption or receptorinteraction of virions, thus preventing infection, 2. doping the virusassembly or the packaging of viral genomes into capsids (competing withviral DNA or RNA for packaging), resulting in defective virions, 3.disrupting and or preventing the formation of capsids during packagingor the interaction of capsid proteins with other structural proteins,resulting in inhibition of viral release or causing the release ofdefective virions, 4. binding to key viral components and preventing orreducing their activity, 5. binding to key host components required forviral proliferation.

[0161] Without being limited on the mechanism by which the present viralinhibition is achieved, as indicated above there are several possiblemechanisms that could explain and/or predict the inhibitory propertiesof ONs against viral replication. The first of these is that the generalaptameric effect of ONs is allowing for their attachment, either toproteins on the cell surface or to viral proteins, preventing viraladsorption and fusion. The size threshold for effect may be a result ofa certain cumulative charge required for interaction.

[0162] A second possible mechanism is that ONs may function within thecell by preventing packaging and/or assembly of the virus. ONs above acertain size threshold may compete or interfere with the normalcapsid/nucleic acid interaction, preventing the packaging of afunctional viral genome inside new viruses. Alternatively, ONs mayprevent the formation of a normal capsid, which could prevent normalviral budding, alter viral stability, or prevent proper viriondisassembly upon internalization.

[0163] While the mechanism of action is not yet entirely clear, assayresults demonstrate that the present ONs can exhibit greater efficacy inviral inhibition compared to the clinical correlates, acyclovir,gancyclovir, Ribavirin, and protease inhibitors. ONs in accordance withthe present invention could thus be used for treating or preventingviral infection. The viral infections treated could be those caused byhuman, animal, and plant viruses

[0164] Broad Spectrum Antiviral Activity

[0165] According to the conclusions discussed above and the datareported herein, it appeared that random ONs and ON randomers could havebroad-spectrum antiviral activity with viruses where assembly and/orpackaging and/or encapsidation of the viral genome is a required step inreplication. Therefore to test this hypothesis, several PS-ODN randomersof different sizes were selected to be tested in cellular models ofvarious viral Infections. A number of such tests are described herein inthe Examples, including tests with CMV, HIV-1, RSV, Coxsackie virus B2,DHBV, Hantavirus, Parainfluenza virus, and Vaccinia virus, as well asthe tests on HSV-1 and HSV-2 described in Examples 1 and 2. Despite thehigh activity level exhibited for some of the tested oligonucleotides,an oligo delivery system such as DOTAP, lipofectamine or oligofectaminecould result in much greater efficacies, especially with the larger (≧40bases) randomers.

[0166] Conclusions on Broad Spectrum Antiviral Activity

[0167] The efficacy studies with different viruses demonstrate thatrandom ONs and randomers display inhibitory properties against a varietyof different viruses. Moreover, these studies support the conclusionthat larger randomers display greater efficacy for viral inhibition thansmaller randomers. This suggests a common size and/or charge dependentmechanism for the random ONs or ON randomers activity in allencapsidating viruses.

[0168] While HSV and CMV are both double-stranded DNA viruses of theherpesviridae family, HIV is a RNA virus from the retroviridae, and RSVa RNA virus from the paramyxoviridae. Given the fact that ON randomerscan inhibit viral function in a variety of different viruses, withoutbeing limited to the mechanisms listed, as discussed above the followingmechanisms are reasonable: A) ONs/ON randomers are inhibiting viralinfection via an aptameric effect, preventing viral fusion with theplasma membrane; and/or B) ONs/ON randomers are preventing or doping theassembly of virions or the packaging of viral DNA within capsidsresulting in defective virions; and/or C)ONs/ON randomers areinterfering with host proteins or components required in the assemblyand/or packaging and/or gene expression of the virus.

[0169] R quirem nt for Antiviral Activity

[0170] Since a randomized DNA sequence seems to be sufficient for viralinhibition, it was interesting to see if antiviral activity could bemaintained in the absence of the phosphorothioate modification and alsoif the efficacy was augmented by either choosing a random sequence or aspecific sequence found in the viral genome.

[0171] Accordingly, DNA and RNA modifications were investigated withrespect to their effect on the antiviral efficacy of the randomers.Since randomers work via a non-sequence complementary mechanism, theseexperiments were designed to test the slight changes in nucleic acidconformation and charge distribution on antiviral efficacy.

[0172] To test if ODNs with different nucleotide/nucleosidemodifications could inhibit HSV-1, REP 2024, 2026, 2059, and 2060 weretested in the HSV-1 PRA as described in the Examples. REP 2024 (a PS-ODNwith a 2-O-Methyl modification to the ribose on 4 bases at both terminiof the ODN), REP 2026 (a PO-ODN with methylphosphonate modifications tothe linkages between the 4 bases at both termini of the ODN), REP 2059(RNA PS-ODN randomer 20 bases in length), and REP 2060 (RNA PS-ODNrandomer 30 bases in length) showed anti-HSV-1 activity (see FIG. 11).

[0173] In the latter example, should the antiviral effect be caused onlyby the ONs consisting of DNA phosphorothioate backbone, such effectwould thus be caused by only one molecule. But other backbones andmodifications gave positive antiviral activity. Of course, one skilledin the art applying the teaching of the present invention could also usedifferent chemistry ONs. A modification of the ON, such as, but notlimited to, a phosphorothioate modification, appears to be beneficialfor antiviral activity. This is most likely due to the needed charge ofONs and/or the requirement for stabilization of DNA both in the mediaand intracellularly, and it may also be due to the chirality of thePS-ODNs.

[0174] Compound REP 2026 showed an antiviral activity while having acentral portion comprising unmodified PO-nucleotides and 4methylphosphonate linkages at both termini protecting from degradation.This indicates that PO-ODNs can be used as antivirals while protectedfrom degradation. This protection can be achieved by modifyingnucleotides at termini and/or by using a suitable delivery system asdescribed later.

[0175] In general, the sequence composition of the DNA used has littleeffect on the overall efficacy, whether randomer, random sequence or aspecific HSV-1 sequence. However, at intermediate lengths, HSV-1sequence was almost 3× more potent than a random sequence (see FIG. 10).This data suggests that while specific antisense functionality existsfor specific HSV sequences, the non-antisense mechanism (non-sequencecomplementary mechanism) elucidated herein may represent the predominantpart of this activity. Indeed, as the ON grows to 40 bases, essentiallyall of the antiviral activity can be attributed to a non-antisenseeffect.

[0176] Lower Toxicity of Randomer

[0177] One goal of using an ON randomer is to lower the toxicity. It isknown that different sequences may trigger different responses in theanimal, such as general toxicity, interaction with serum proteins, andinteraction with immune system (Monteith et al (1998) Toxicol Sci46:365-375). The mixture of ONs may thus decrease toxic effects becausethe level of any particular sequence will be very low, so that nosignificant interaction due to sequence or nucleotide composition islikely.

[0178] Pharmaceutical Compositions

[0179] The ONs of the invention may be in the form of a therapeuticcomposition or formulation useful for treating (or prophylaxis of) viraldiseases, which can be approved by a regulatory agency for use in humansor in non-human animals, and/or against a particular virus or group ofviruses. These ONs may be used as part of a pharmaceutical compositionwhen combined with a physiologically and/or pharmaceutically acceptablecarrier. The characteristics of the carrier may depend on the route ofadministration. The pharmaceutical composition of the invention may alsocontain other active factors and/or agents which enhance activity.

[0180] Administration of the ONs of the invention used in thepharmaceutical composition or formulation or to practice the method oftreating an animal can be carried out in a variety of conventional ways,such as intraocular, oral ingestion, enterally, inhalation, orcutaneous, subcutaneous, intramuscular, intraperitoneal, intrathecal,intratracheal, or intravenous injection.

[0181] The pharmaceutical composition or oligonucleotide formulation ofthe invention may further contain other chemotherapeutic drugs for thetreatment of viral diseases, such as, without limitation, Rifampin,Ribavirin, Pleconaryl, Cidofovir, Acyclovir, Pencyclovir, Gancyclovir,Valacyclovir, Famciclovir, Foscarnet, Vidarabine, Amantadine, Zanamivir,Oseltamivir, Resquimod, antiproteases, HIV fusion inhibitors, nucleotideHIV RT inhibitors (e.g., AZT, Lamivudine, Abacavir), non-nucleotide HIVRT inhibitors, Doconosol, Interferons, Butylated Hydroxytoluene (BHT)and Hypericin. Such additional factors and/or agents may be included inthe pharmaceutical composition, for example, to produce a synergisticeffect with the ONs of the invention.

[0182] The pharmaceutical composition or oligonucleotide formulation ofthe invention may further contain a polymer, such as, withoutrestriction, polyanionic agents, sulfated polysaccharides, heparin,dextran sulfate, pentosan polysulfate, polyvinylalcool sulfate,acemannan, polyhydroxycarboxylates, cellulose sulfate, polymerscontaining sulfonated benzene or naphthalene rings and naphthalenesulfonate polymer, acetyl phthaloyl cellulose, poly-L-lysine, sodiumcaprate, cationic amphiphiles, cholic acid. Polymers are known to affectthe entry of virions in cells by, in some cases, binding or adsorbing tothe virion itself. This characteristic of antiviral polymers can beuseful in competing with ONs for the binding, or adsorption to thevirion, the result being an increased intracellular activity of the ONscompared to its extracellular activity.

[0183] Ex mplary D liv ry System

[0184] We monitored the uptake of PS-ODN randomers by exposing culturedcells to fluorescently labeled randomers and then examined thefluorescence intensity in lysed cells after two rounds of washing. Thecellular uptake of cells exposed to 250 nM REP 2004-FL was tested withno delivery and after encapsulation in one of the following lipid baseddelivery systems; Lipofectamine™ (Invitrogen), Polyfect™ (Qiagen) andOligofectamine™ (Invitrogen). After 4 hours, cells were washed twicewith PBS and lysed using MPER lysis reagent (PROMEGA). FIG. 34 shows therelative fluorescence yield from equivalent numbers of exposed cellswith and without delivery. We observe than in the presence of all threedelivery agents tested, there was a significant increase in theintracellular PS-ODN concentration compared to no delivery.

[0185] In keeping with the test results, the use of a delivery systemcan significantly increase the antiviral potency of ON randomers.Additionally, they will serve to protect these compounds from seruminteractions, reducing side effects and maximizing tissue and cellulardistribution.

[0186] Although PS-ODNs are more resistant to endogenous nucleases thannatural phosphodiesters, they are not completely stable and are slowlydegraded in blood and tissues. A limitation in the clinical applicationof PS oligonucleotide drugs is their propensity to activate complementon i.v. administration. In general, liposomes and other delivery systemsenhance the therapeutic index of drugs, including ONs, by reducing drugtoxicity, increasing residency time in the plasma, and delivering moreactive drug to disease tissue by extravasation of the carriers throughhyperpermeable vasculature. Moreover in the case of PS-ODN, lipidencapsulation prevents the interaction with potential protein-bindingsites while in circulation (Klimuk et al. (2000) J Pharmacol Exp Ther292:480-488).

[0187] According to our results described herein, an approach is to usea delivery system such as, but without restriction, lipophilicmolecules, polar lipids, liposomes, monolayers, bilayers, vesicles,programmable fusogenic vesicles, micelles, cyclodextrins, PEG,iontophoresis, powder injection, and nanoparticles (such as PIBCA,PIHCA, PHCA, gelatine, PEG-PLA) for the delivery of ONs describedherein. The purpose of using such delivery systems are to, among otherthings, lower the toxicity of the active compound in animals and humans,increase cellular delivery, lower the IC50, increase the duration ofaction from the standpoint of drug delivery and protect theoligonucleotides from non-specific binding with serum proteins.

[0188] We have shown that the antiviral activity of PS-ODN randomersincreases with increasing size. Moreover this activity is correlatedwith increased affinity for viral proteins (in a viral lysate). Since itis well known in the art that the phosphorothioate modificationincreases the affinity of protein-DNA interaction, we tested the abilityof increasingly larger PS-ODN randomers to bind to fetal bovine serum(FBS) (FIG. 28a) using the same FP-based assay used for measuringinteraction with viral lysates. In this assay, 250 ug of non-heatinactivated FBS was complexed with a fluorescently labeled 20 basePS-ODN randomer, under conditions where the binding (mP value) wassaturated. Unlabelled PS-ODN randomers of increasing size (REP 2003, REP2004, REP 2006 and REP 2007) were used to compete the interaction of FBSwith the labeled bait. The results of this test clearly show that as thesize of the PS-ODN randomer increases, so does its affinity for FBS.This result suggests that the most highly active anti-viral PS-ODNs willalso be the ones to bind with the highest affinity to proteins.

[0189] It is known in the art that one of the main therapeutic problemsfor phosphorothioate antisense oligonucleotides is their side effectsdue mainly to this increased interaction with proteins (specificallywith serum proteins) as described by Kandimalla and co-workers(Kandimalla et al. (1998) Bioorg. Med. Chem. Lett. 8:2103-2108). Ourdata suggests substantial benefits by a suitable delivery system capableof delivering antiviral ONs into the cell while preventing theirinteraction with serum proteins.

[0190] To demonstrate the benefits of a delivery system, we tested twodifferent delivery technologies which are liposomal based; Cytofectinand DOTAP. We measured the delivery of the PS-ODN randomer REP 2006(encapsulated with either Cytofectin or DOTAP) into 293A cells in thepresence of high concentrations of serum (50%) by measuring theintracellular concentration of labeled REP 2006 by fluorometry (FIGS.28b, c). These results show that delivery increases the intracellularconcentration of REP 2006, and also that, in the case of DOTAP, thelevels of intracellular REP 2006 after 24 hours were markedly increased.Finally, we measured the protection of REP2006 from serum proteininteractions by DOTAP (28d) and cytofectin (28e) in our in vitroFP-based interaction assay. Unencapsulated REP 2006 was able to competebound fluorescent oligo from serum but when REP 2006 was encapsulatedwith either DOTAP or cytofectin it was no longer able to compete forserum binding. These data suggest that encapsulation protects oligosfrom serum interaction and will result in a more effective therapeuticeffect with fewer side effects.

[0191] Another potential benefit in using a delivery system is toprotect the ONs from interactions, such as adsorption, with infectivevirions in order to prevent amplification of viral infection throughdifferent mechanisms such as increased cellular penetration of virions.

[0192] Another approach is to accomplish cell specific delivery byassociating the delivery system with a molecule(s) that will increaseaffinity with specific cells, such molecules being without restrictionantibodies, receptor ligands, vitamins, hormones and peptides.

[0193] Additional options for delivery systems are provided below.

[0194] Linked ODN

[0195] In certain embodiments, ONs of the invention are modified in anumber of ways without compromising their ability to inhibit viralreplication. For example, the ONs are linked or conjugated, at one ormore of their nucleotide residues, to another moiety. Thus, modificationof the oligonucleotides of the invention can involve chemically linkingto the oligonucleotide one or more moieties or conjugates which enhancethe activity, cellular distribution or cellular uptake, increasetransfer across cellular membranes specifically or not, or protectingagainst degradation or excretion, or providing other advantageouscharacteristics. Such advantageous characteristics can, for example,include lower serum interaction, higher viral-protein interaction, theability to be formulated for delivery, a detectable signal, improvedpharmacokinetic properties, and lower toxicity. Such conjugate groupscan be covalently bound to functional groups such as primary orsecondary hydroxyl groups. For example, conjugate moieties can include asteroid molecule, a non-aromatic lipophilic molecule, a peptide,cholesterol, bis-cholesterol, an antibody, PEG, a protein, a watersoluble vitamin, a lipid soluble vitamin, another ON, or any othermolecule improving the activity and/or bioavailability of ONs.

[0196] In greater detail, exemplary conjugate groups of the inventioncan include intercalators, reporter molecules, polyamines, polyamides,polyethylene glycols, polyethers, SATE, t-butyl-SATE, groups thatenhance the pharmacodynamic properties of oligomers, and groups thatenhance the pharmacokinetic properties of oligomers. Typical conjugategroups include cholesterols, lipids, phospholipids, biotin, phenazine,folate, phenanthridine, anthraquinone, acridine, fluoresceins,rhodamines, coumarins, fluorescent nucleobases, and dyes.

[0197] Groups that enhance the pharmacodynamic properties, in thecontext of this invention, include groups that improve oligomer cellularuptake and/or enhance oligomer resistance to degradation and/or protectagainst serum interaction. Groups that enhance the pharmacokineticproperties, in the context of this invention, include groups thatimprove oligomer uptake, distribution, metabolism or excretion.Exemplary conjugate groups are described in International PatentApplication PCT/US92/09196, filed Oct. 23, 1992, which is incorporatedherein by reference in its entirety.

[0198] Conjugate moieties can include but are not limited to lipidmoieties such as a cholesterol moiety (Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al.,Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,306-309; Manoharan et al., Bioorg. Med. Chem. Let, 1993, 3, 2765-2770),a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20,533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues(Saison-Behmoaras et at., EMBO J., 1991, 10,1111-1118; Kabanov et al.,FEBS Lett., 1990, 259, 327-330; Svinarchuk et at., Biochimie, 1993, 75,49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et at., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et at., Nucleosides & Nucleotides,1995,14,969-973), or adamantane acetic acid (Manoharan et at.,Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra etat., Biochim. Biophys. Acta, 1995,1264,229-237), or an octadecylamine orhexylaminocarbonyl-oxycholesterol moiety (Crooke et al., J. PharmacolExp. Ther., 1996, 277, 923-937.

[0199] The present oligonucleotides may also be conjugated to activedrug substances, for example without limitation, aspirin, warfarin,phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen,(S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoicacid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide,a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug,an antidiabetic, an antibacterial or an antibiotic.

[0200] Exemplary U.S. patents that describe the preparation of exemplaryoligonucleotide conjugates include, for example, U.S. Pat. Nos.4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of whichis incorporated by reference herein in its entirety.

[0201] Another approach is to prepare antiviral ONs as lipophilicpro-oligonucleotides by modification with enzymatically cleavable chargeneutralizing adducts such as s-acetylthio-ethyl or s-pivasloylthio-ethyl(Vives et al., 1999, Nucl Acids Res 27: 4071-4076). Such modificationshave been shown to increase the uptake of ONs into cells.

[0202] Design of Non-Specific ONs

[0203] In another approach, an antiviral ON demonstrating low,preferably the lowest possible, homology with the human (or othersubject organism) genome is designed. The goal is to obtain an ON thatwill show the lowest toxicity due to interactions with human or animalgenome sequence(s) and mRNAs. The first step is to produce the desiredlength sequence of the ON, e.g., by aligning nucleotides A, C, G, T in arandom fashion, manually or, more commonly, using a computer program.The second step is to compare the ON sequence with a library of humansequences such as GenBank and/or the Ensemble Human Genome Database. Thesequence generation and comparison can be performed repetitively, ifdesired, to identify a sequence or sequences having a desired lowhomology level with the subject genome. Desirably, the ON sequence is atthe lowest homology possible with the entire genome, while alsopreferably minimizing self interaction.

[0204] Non-Specific ONs With Antisense Activity

[0205] In another approach, an antiviral non-specific sequenceportion(s) is/are coupled with an antisense sequence portion(s) toincrease the activity of the final ON. The non-specific portion of theONs is described in the present invention. The antisense portion iscomplementary to a viral mRNA.

[0206] Non-Specific ONs with a G-Rich Motif Activity

[0207] In another approach, an antiviral non-specific sequenceportion(s) is/are coupled with a motif portion(s) to improve theactivity of the final ON. The non-specific portion of the ON isdescribed in the present invention. The motif portion can, asnon-limiting examples, include, CpG, Gquartet, and/or CG that aredescribed in the literature as stimulators of the immune system. Agrawalet al. (2001) Curr. Cancer Drug Targets 3:197-209.

[0208] Non-Watson-Crick ONs

[0209] Another approach is to use an ON composed of one type or more ofnon-Watson-Crick nucleotides/nucleosides. Such ONs can mimic PS-ODNswith some of the following characteristics similar to PS-ODNs: a) thetotal charge; b) the space between the units; c) the length of thechain; d) a net dipole with accumulation of negative charge on one side;e) the ability to bind to proteins; f) the ability to bind viralproteins, g) the ability to penetrate cells, h) an acceptabletherapeutic index, i) an antiviral activity. The ON has a preferredphosphorothioate backbone but is not limited to it. Examples ofnon-Watson-Crick nucleotides/nucleosides are described in Kool, 2002,Acc. Chem. Res. 35:936-943; and Takeshita et al., (1987) J. Biol. Chem.262:10171-10179 where ODNs containing synthetic abasic sites aredescribed.

[0210] Antiviral Polymer

[0211] Another approach is to use a polymer mimicking the activity ofphophorothioate ODNs. As described in the literature, several anionicpolymers were shown to have antiviral inhibitory activity. Thesepolymers belong to several classes: (1) sulfate esters ofpolysaccharides (dextrin and dextran sulfates; cellulose sulfate); (2)polymers containing sulfonated benzene or naphthalene rings andnaphthalene sulfonate polymers; (3) polycarboxylates (acrylic acidpolymers); and acetyl phthaloyl cellulose (Neurath et al. (2002) BMCInfect Dis 2:27); and (4) abasic oligonucleotides (Takeshita et al.,1987, J. Bio. Chem. 262:10171-10179). Other examples of non-nucleotideantiviral polymers are described in the literature. The polymersdescribed herein mimic PS-ODNs described in this invention and have thefollowing characteristics similar to PS-ODNs: a) the length of thechain; b) a net dipole with accumulation of negative charge on one side;c) the ability to bind to proteins; d) the ability to bind viralprotein, e) an acceptable therapeutic index, f) an antiviral activity.In order to mimic the effect of a PS-ODN, the antiviral polymer maypreferably be a polyanion displaying similar space between its units ascompared to a PS-ODN. It may also have the ability to penetrate cellsalone or in combination with a delivery system.

[0212] Antiviral Activity of Double-Stranded PS-ODNs

[0213] A random sequence (REP 2017) and its complement (either PSmodified or unmodified) are fluorescently labeled as described elsewhereand tested for their ability to bind to purified HSV-1 and HIV-1proteins by fluorescence polarization as described in the presentinvention. Hybridization was verified by acrylamide gel electrophoresis.Unmodified REP 2017 (2017U), either single (ss) or double stranded (ds),had no binding activity in either HSV-1 or HIV-1 lysates. However, PSmodified REP 2017, either single stranded or double stranded, wascapable of HSV-1 and HIV-1 interaction (see FIG. 33).

[0214] According to our results described herein, an approach is to usedouble stranded ONs as effective antiviral agents. Preferentially suchONs have a phosphorothioate backbone but may also have other and/oradditional modifications which increase either their delivery and/orantiviral activity and/or stability as described herein for singlestranded ONs.

[0215] In Vitro Assay for Drug Discovery

[0216] An in vitro assay is developed based on fluorescence polarizationto measure the ability of PS-ODNs to bind to viral components, e.g.,viral proteins. When a protein (or another interactor) binds to thefluorescently labeled bait, the three dimensional tumbling of the baitin solution is retarded. The retardation of this tumbling is measured byan inherent increase in the polarization of excited light from thelabeled bait. Therefore, increased polarization (reported as adimensionless measure [mP]) is correlated with increased binding.

[0217] One methodology is to use as bait a PS-ODN randomer labeled atthe 3′ end with FITC using an inflexible linker (3′-(6-Fluorescein)CPG). This PS-ODN randomer is diluted to 2 nM in assay buffer (10 mMTris, pH7.2, 80 mM NaCl, 10 mM EDTA, 100 mM b-mercaptoethanol and 1%tween 20). This oligo is then mixed with an appropriate interactor. Inthis case, we use lysates of sucrose gradient purified HSV-1 (strainMacIntyre), HIV-1 (strain Mn) or RSV (strain A2) suspended in 0.5M KCland 0.5% Triton X-100 (HSV-1 and HIV-1) or 10 mM Tris, pH7.5, 150 mMNaCl, 1 mM EDTA and 0.1% Triton X-100 (RSV). Following bait interaction,the complexes are challenged with various unlabelled PS-ODNs to assesstheir ability to displace the bait from its complex.

[0218] In FIG. 29, we show a preliminary test with three baits ofdifferent sizes; 6 (REP 2032-FL), 10 (REP 2003-FL) and 20 bases (REP2004-FL). These baits were tested for their ability to interact withHSV-1 (FIG. 29a), HIV-1 (FIG. 29b) and RSV (FIG. 29c) lysates. In thepresence of any of the viral lysates the degree of binding was dependenton the size of the bait used, with 2004-FL displaying the largest shiftin mP (binding) in the presence of viral lysate. We note that this issimilar to the size dependent antiviral efficacy of PS-ODN randomers.This bait was then used to assess the ability of PS-ODNs of differentsizes to compete the interaction of the bait with the lysate.

[0219] In FIG. 30, the interaction of REP 2004-FL with HSV-1 (FIG. 30a),HIV-1. (FIG. 30b) and RSV (FIG. 30c) lysates is challenged with PS-ODNsof increasing size. For each viral lysate tested, we note that REP 2003is unable to compete the bait away from the lysate. The bait interactionwas very strong as revealed by the relatively weak competition elicitedby the REP 2004 (unlabeled bait) competitor. However, it was observedthat as the size of the competitor PS-ODN increased above 20 bases, itsability to displace the bait became more robust. This indicates anincreased affinity to protein components in the viral lysate as thePS-ODN randomer size increases. This phenomenon mirrors the increasedantiviral activity of larger PS-ODN randomers against HSV-1, HSV-2, CMV,HIV-1 and RSV.

[0220] The similarity between the efficacy in bait competition andantiviral activity of PS-ODN randomers indicates that this assayparadigm is a good predictor of antiviral activity. This assay isrobust, easy to perform and very stable, making it a very good candidatefor a high throughput screen to identify novel antiviral molecules basednot on specific target identification but on their ability to interactwith one or more components, e.g., viral proteins.

[0221] While the exemplary method described herein utilizes fluorescencepolarization to measure interaction with the viral lysate, numeroustechniques are known in the art for monitoring protein interactions, andcan be used in the present methods. These include without restrictionsurface plasmon resonance, fluorescence resonance energy transfer(FRET), enzyme linked immunosorbent assay (ELSIA), gel electrophoresis(to measure mobility shift), isothermal titration and differentialscanning microcalorimetry and column chromatography. These otherdifferent techniques can be applied to measure the interaction of ONswith a viral lysate or component, and thus can be useful in screeningfor compounds which have anti-viral activity.

[0222] The method described herein is used to screen for novel compoundsfrom any desired source, for example, from a library synthesized bycombinatorial chemistry or isolated by purification of naturalsubstances. It can be used to a) determine appropriate size,modifications, and backbones of novel ONs; b) test novel moleculesincluding novel polymers; predict a particular virus' susceptibility tonovel ONs or novel compounds; or d) determine the appropriate suite ofcompounds to maximally inhibit a particular virus.

[0223] The increased lysate affinity with larger sized PS-ODN randomerssuggests that the antiviral mechanism of action of PS-ODN randomers isbased on an interaction with one or more viral protein components whichprevents either the infection or correct replication of virions. It alsosuggests that this interaction is charge (size) dependent and notdependent on sequence. As these PS-ODN randomers have a size dependentactivity across multiple viruses spanning several different families, wesuggest that PS-ODN randomers interfere with common, charge dependentprotein-protein interactions, protein-DNA/RNA interactions, and/or othermolecule-molecule interactions. These interactions can include (but arenot limited to):

[0224] a. The interaction between individual capsid subunits duringcapsid formation.

[0225] b. The interaction between the capsid/nucleocapsid protein andthe viral genome.

[0226] c. The interaction between the capsid and glycoproteins duringbudding.

[0227] d. The interaction between the glycoprotein and its receptorduring infection.

[0228] e. The interaction between other viral key components involved inviral replication.

[0229] These multiple, simultaneous inhibitions of protein-proteininteractions represent a novel mechanism for antiviral inhibition.

[0230] Effect of PS-ODN Sequence Composition n Lysate

[0231] We monitored the ability of PS-ODNs of different sequences tointeract with several viral lysates. In each case, a 20-mer PS-ODN islabeled at the 3′ end with FITC as previously described herein. ThePS-ODNs tested consisted of A20, T20, G20, C20, AC10, AG10, TC10, TG10,REP 2004 and REP 2017. Each of these sequences is diluted to 4 nM inassay buffer and incubated in the presence of lug of HSV-1, HIV-1 or RSVlysate. Interaction is measured by fluorescence polarization.

[0232] The profile of interaction with all sequences tested is similarin all viral lysates, indicating that the nature of the bindinginteraction is very similar. Within each lysate, the PS-ODNs of uniformcomposition (A20, G20, T20, C20) were the weakest interactors with A20being the weakest interactor of these by a significant margin. For therest of the PS-ODNs tested, all of them displayed a similar, stronginteraction with the exception of TG10, which consistently displayed thestrongest interaction in each lysate (see FIG. 35). Targetidentification for PS-ODN randomers in HIV-1

[0233] The ability of PS-ODN randomers to bind to purified HIV-1proteins is tested by fluorescence polarization as described in example9. Increasing quantities of purified HIV-1 p24 or purified HIV-1 gp41were reacted with REP 2004-FL (see FIG. 31). We note that for both theseproteins, there is a protein concentration dependent shift influorescence polarization, indicating an interaction with both theseproteins.

[0234] The ability of a range of sizes of PS-ODN randomers to bind tothese proteins was also tested using fluorescent versions of REP 2032,REP 2003, REP 2004, REP 2006 and REP 2007 (see FIG. 32). We observe thatfor p24, there is no size dependent interaction with p24 (see FIG. 32a)however; we did see an increase in gp41 binding in PS-ODN randomerslarger than 20 bases versus those less than 20 bases (see FIG. 32b).This suggests when PS-ODN randomer length increases above 20 bases,multiple copies of gp41 can bind to individual randomers, increasingtheir polarization.

[0235] This is a significant observation as it demonstrates thepotential of larger ONs to sequester structural proteins during viralsynthesis and limit their availability for the formation of new virions.

[0236] High Affinity Oligonucleotides

[0237] Another approach is a method to enrich or purify antiviral ON(s)having a higher affinity for viral components, such as viral proteins,than the average affinity of the ONs in a starting pool of ONs. Themethod will thus provide one or more non-sequence complementary ON(s)that will exhibit increased affinity to one or more viral components,e.g., having a three-dimensional shape contributing to such elevatedbinding affinity. The rationale is that while ON(s) will act as linearmolecules in binding with viral components, they can also fold into a3-dimensional shape that can enhance the interaction with such viralcomponents. Without being limited to the specific technique, highaffinity ONs can be purified or enriched in the following ways.

[0238] One method for purifying a high affinity ON, or a plurality ofhigh affinity ONs, involves using a stationary phase medium with boundviral protein(s) as an affinity matrix to bind ONs, which can then beeluted under increasingly stringent conditions (e.g., increasingconcentration of salt or other chaotropic agent, and/or increasingtemperture and/or changes in pH). Such a method can, for example, becarried out by:

[0239] (a) loading a pool of ONs onto an exchange column having a viralprotein or several viral proteins or a viral lysate bound to astationary phase;

[0240] (b) displacing (eluting) bound ONs from the column, e.g., byusing a displacer solution such as an increasing salt solution;

[0241] (c) collecting fractions of eluted ONs at different saltconcentration;

[0242] (d) cloning and sequencing eluted ONs from different fractions,more preferably from a fraction(s) at high salt concentration, such thatthe ONs eluted at the high salt concentration have a greater bindingaffinity with the viral protein(s); and

[0243] (e) Testing the activity of sequenced ON(s) in assays suchbinding and/or viral inhibition assay, e.g., a fluorescencepolarization-binding assay as decribed herein and/or in a cellular viralinhibition assay and/or in an animal viral inhibition assay.

[0244] In a second example, a method derived and modified from the SELEXmethodology (Morris et al (1998) Biochemistry 95:2902-2907) can be usedfor purifying the high affinity ON. One implementation of such a methodcan be performed as:

[0245] (a) providing a starting ON pool material, for example, acollection of synthetic random ONs containing a high number ofsequences, e.g., one hundred trillion (1014) to ten quadrillion (1016)different sequences. Each ON molecule contains a segment of randomsequence flanked by primer-binding sequences at each end to facilitatepolymerase chain reaction (PCR). Because the nucleotide sequences ofessentially all of the molecules are unique, an enormous number ofstructures are sampled in the population. These structures determineeach molecule's biochemical properties, such as the ability to bind agiven viral target molecule;

[0246] (b) contacting ONs with a viral protein or several viral proteinsor a viral lysate;

[0247] (c) selecting ONs that bind to viral protein(s), using apartition technique(s) that can partition bound and unbound ONs, such asnative gel shifts and nitrocellulose filtration. Either of these methodsphysically separates the bound species from the unbound species,allowing preferential recovery of those sequences that bind best. Also,to select ON (s) that bind to a small protein, it is desirable to attachthe target to a solid support and use that support as an affinitypurification matrix. Those molecules that are not bound get washed offand the bound ones are eluted with free target, again physicallyseparating bound and unbound species;

[0248] (d) amplifying the eluted binding ON(s), e.g., by using PCR usingprimers hybridizing with both flanking sequences of ONs;

[0249] (e) steps (b) (c) and (d) can be performed multiple times (i.e.,multiple cycles or rounds of enrichment and amplification) in order topreferentially recover ONs that display the highest binding affinity toviral protein(s). After several cycles of enrichment and amplification,the population is dominated by sequences that display the desiredbiochemical property;

[0250] (f) cloning and sequencing one or more ONs selected from from anenrichment cycle, e.g., the last such cycle; and

[0251] (g) testing the binding and/or activity of sequenced ON(s) inassays, e.g., in a fluorescence polarization binding assay as decribedherein and/or in a cellular viral inhibition assay and/or in an animalviral inhibition assay.

[0252] Another approach is to apply a modification of a split synthesismethodology to create one-bead one-PS-ODN and one-bead one-PS2—ODNlibraries as described in Yang et al (2002) Nucl. Acids Res.30(e132):1-8. Binding and selection of specific beads to viral proteinscan be done. Sequencing both the nucleic acid bases and the positions ofany thioate/dithioate linkages can be carried out by using a PCR-basedidentification tag of the selected beads. This approach can allow forthe rapid and convenient identification of PS-ODNs or PS2-ODNs that bindto viral proteins and that exhibit potent antiviral properties.

[0253] Once the specific sequences that bind to the viral proteins withhigh affinity are determined (e.g., by amplification and sequencing ofindividual sequences), one or more such high affinity sequences can beselected and synthesized (e.g., by either chemical or enzymaticsynthesis) to provide a preparation of high affinity ON(s), which can bemodified to improve their activity, including improving theirpharmacokinetic properties. Such high affinity ONs can be used in thepresent invention.

[0254] Prion Diseases

[0255] Another approach is used in an alternative embodiment of thepresent invention for the treatment, the control of the progression, orthe prevention of prion disease. This fatal neurodegenerative disease isinfectious and can affect both humans and animals. Structural changes inthe cellular prion protein, PrPC to its scrapie isoform, PrPSC, areconsidered to be the obligatory step in the occurrence and propagationof the prion disease. Amyloid polymers are associated withneuropathology of the prion disease.

[0256] The incubation of a prion protein fragment and double strandednucleic acid results in the formation of amyloid fibres (Nandi et al(2002), J Mol Biol 322: 153-161). ONs having affinity to proteins suchas phosphorothioates are used to compete or inhibit the interaction ofdouble stranded nucleic acid with the PrPC and consequently stop theformation of the amyloid polymers. Such ONs of different sizes anddifferent compositions can be used in an appropriate delivery form totreat patients suffering from prion diseases or for prophylaxis in highrisk situations. Such interfering ONs can be identified by measuringfolding changes of amyloid polymerase as described by Nandi et al.(supra) in the presence of test ONs.

[0257] Putative Viral Etiologies

[0258] Another approach is used in another embodiment of the presentinvention for the treatment or prevention of diseases or conditions withputative viral etiologies as described without limitation in thefollowing examples. Viruses are putative causal agents in diseases andconditions that are not related to a primary viral infection. Forexample, arthritis is associated with HCV (Olivieri et al. (2003) RheumDis Clin North Am 29:111-122), Parvovirus B19, HIV, HSV, CMV, EBV, andVZV (Stahl et al. (2000) Clin Rheumatol 19:281-286). Other viruses havealso been identified as playing a role in different diseases. Forexample, influenza A in Parkinson's disease (Takahashi et al. (1999),Jpn J Infect Dis 52:89-98), Coronavirus, EBV and other viruses inMultiple Sclerosis (Talbot et al (2001) Curr Top Microbiol Immunol253:247-71); EBV, CMV and HSV-6 in Chronic Fatigue Syndrome (Lerner etal. (2002) Drugs Today 38:549-561); and paramyxoviruses in asthma(Walter et al (2002) J Clin Invest 110:165-175) and in Paget's disease;and HBV, HSV, and influrenza in Guillain-Barre Syndrome.

[0259] Because of these etiologies, inhibition of the relevant virususing the present invention can delay, slow, or prevent development ofthe corresponding disease or condition, or at least some symptoms ofthat disease.

[0260] Oligonucleotide Modifications and Synthesis

[0261] As indicated in the Summary above, modified oligonucleotides areuseful in this invention. Such modified oligonucleotides include, forexample, oligonucleotides containing modified backbones or non-naturalinternucleoside linkages. Oligonucleotides having modified backbonesinclude those that retain a phosphorus atom in the backbone and thosethat do not have a phosphorus atom in the backbone.

[0262] Such modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotri-esters, methyl and other alkylphosphonates including 3′-alkylene phosphonates, 5′-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidatesincluding 3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates, carboranyl phosphate andborano-phosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Oligonucleotides having inverted polarity typically include a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

[0263] Preparation of oligonucleotides with phosphorus-containinglinkages as indicated above are described, for example, in U.S. Pat.Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897;5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676;5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126;5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361;5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050,each of which is incorporated by reference herein in its entirety.

[0264] Some exemplary modified oligonucleotide backbones that do notinclude a phosphorus atom have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts. Particularlyadvantageous are backbone linkages that include one or more chargedmoieties. Examples of U.S. patents describing the preparation of thepreceding oligonucleotides include U.S. Pat. Nos. 5,034,506; 5,166,315;5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564;5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489, 677; 5,541,307;5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437;5,792,608; 5,646,269 and 5,677,439, each of which is incorporated byreference herein in its entirety.

[0265] Modified oligonucleotides may also contain one or moresubstituted sugar moieties. For example, such oligonucleotides caninclude one of the following 2′-modifications: OH; F; O-, S-, orN-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl,wherein the alkyl, alkenyl and alkynyl may be substituted orunsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl, or2′-O—(O-carboran-1-yl)methyl. Particular examples areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)—OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON [(CH₂)_(n)CH₃)]₂, where n and m arefrom 1 to 10. Other exemplary oligonucleotides include one of thefollowing 2′-modifications: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH₃, OCN, Cl, Br, CN, CF₃. OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide. Examples include 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃,also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv.Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group;2′-dimethy-laminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE; and 2′-dimethylaminoethoxyethoxy (also known as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂.

[0266] Other modifications include Locked Nucleic Acids (LNAS) in whichthe 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugarring thereby forming a bicyclic sugar moiety. The linkage can be amethelyne (—CH₂—)˜ group bridging the 2′ oxygen atom and the 4′ carbonatom wherein n is 1 or 2. LNAs and preparation thereof are described inWO 98/39352 and WO 99/14226, which are incorporated herein by referencein their entireties.

[0267] Other modifications include sulfur-nitrogen bridge modifications,such as locked nucleic acid as described in Orum et al. (2001) Curr.Opin. Mol. Ther. 3:239-243.

[0268] Other modifications include 2′-methoxy (2′-O—CH₃),2′-methoxyethyl (2′O—CH₂—CH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂),2′-allyl (2′-CH—CH═CH₂), 2′-O-allyl (2′-O—CH₂—CH═CH₂) and 2′-fluoro(2′-F). The 2′-modification may be in the arabino (up) position or ribo(down) position. Similar modifications may also be made at otherpositions on the oligonucleotide, particularly the 3′ position of thesugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotidesand the 5′ position of the 5′ terminal nucleotide. Oligonucleotides mayalso have sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Exemplary U.S. patents describing the preparationof such modified sugar structures include, for example, U.S. Pat. Nos.4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393, 878; 5,446,137;5,466,786; 5,514,785; 5,519,134; 5,567, 811; 5,576,427; 5,591,722;5,597,909; 5,610,300; 5,627, 053; 5,639,873; 5,646,265; 5,658,873;5,670,633; 5,792, 747; and 5,700,920, each of which is incorporated byreference herein in its entirety.

[0269] Still other modifications include an ON concatemer consisting ofmultiple oligonucleotide sequences joined by a linker(s). The linkermay, for example, consist of modified nucleotides or non-nucleotideunits. In some embodiments, the linker provides flexibility to the ONconcatemer. Use of such ON concatemers can provide a facile method tosynthesize a final molecule, by joining smaller oligonucleotide buildingblocks to obtain the desired length. For example, a 12 carbon linker(C12 phosphoramidite) can be used to join two or more ON concatemers andprovide length, stability, and flexibility.

[0270] As used herein, “unmodified” or “natural” bases (nucleobases)include the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C) and uracil (U). Oligonucleotides mayalso include base modifications or substitutions. Modified bases includeother synthetic and naturally-occurring bases such as 5-methylcytosine(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil andcytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynylderivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine,5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines,5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituteduracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Additionalmodified bases include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazi n-2(3H)-one), carbazole cytidine(2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified bases mayalso include those in which the purine or pyrimidine base is replacedwith other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine,2-aminopyridine and 2-pyridone. Further nucleobases include thosedescribed in U.S. Pat. No. 3,687,808, those disclosed in The ConciseEncyclopedia Of Polymer Science And Engineering, pages 858-859,Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed byEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993.

[0271] Another modification includes phosphorodithioate linkages.Knowing that phosphorodithioate ODNs (PS2-ODNs) and PS-ODNs have asimilar binding affinity to proteins (Tonkinson et al. (1994) AntisenseRes. Dev. 4:269-278)(Cheng et al. (1997) J. Mol. Recogn. 10:101-107) andknowing that a possible mechanism of action of ODNs is binding to viralproteins, it could be desirable to include phosphorodithioate linkageson the antiviral ODNs described in this invention.

[0272] Another approach to modify ODNs is to produce stereodefined orstereo-enriched ODNs as described in Yu at al (2000) Bioorg. Med. Chem.8:275-284 and in Inagawa et al. (2002) FEBS Lett. 25:48-52. ODNsprepared by conventional methods consist of a mixture of diastereomersby virtue of the asymmetry around the phosphorus atom involved in theinternucleotide linkage. This may affect the stability of the bindingbetween ODNs and viral components such as viral proteins. Previous datashowed that protein binding is significantly stereo-dependent (Yu etal.). Thus, using stereodefined or stereo-enriched ODNs could improvetheir protein binding properties and improve their antiviral efficacy.

[0273] The incorporation of modifications such as those described abovecan be utilized in many different incorporation patterns and levels.That is, a particular modification need not be included at eachnucleotide or linkage in an oligonucleotide, and different modificationscan be utilized in combination in a single oligonucleotide, or even in asingle nucleotide.

[0274] Oligonucleotide Synthesis

[0275] The present oligonucleotides can by synthesized using methodsknown in the art. For example, unsubstituted and substitutedphosphodiester (P═O) oligonucleotides can be synthesized on an automatedDNA synthesizer (e.g., Applied Biosystems model 380B) using standardphosphoramidite chemistry with oxidation by iodine. Phosphorothioates(P═S) can be synthesized as for the phosphodiester oligonucleotidesexcept the standard oxidation bottle can be replaced by 0.2 M solutionof 311-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for thestep-wise thioation of the phosphite linkages. The thioation wait stepcan be increased to 68 sec, followed by the capping step. After cleavagefrom the CPG column and deblocking in concentrated ammonium hydroxide at55° C. (18 h), the oligonucleotides can be purified by precipitatingtwice with 2.5 volumes of ethanol from a 0.5 M NaCl solution.

[0276] Phosphinate oligonucleotides can be prepared as described in U.S.Pat. No. 5,508,270; alkyl phosphonate oligonucleotides can be preparedas described in U.S. Pat. No. 4,469,863; 3′-Deoxy-3′-methylenephosphonate oligonucleotides can be prepared as described in U.S. Pat.Nos. 5,610,289 and 5,625,050; phosphoramidite oligonucleotides can beprepared as described in U.S. Pat. No. 5,256,775 and U.S. Pat. No.5,366,878; alkylphosphonothioate oligonucleotides can be prepared asdescribed in published PCT applications PCT/US94/00902 andPCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively);3′-Deoxy-3′-amino phosphoramidate oligonucleotides can be prepared asdescribed in U.S. Pat. No. 5,476,925; Phosphotriester oligonucleotidescan be prepared as described in U.S. Pat. No. 5,023,243; boranophosphate oligonucleotides can be prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198; methylenemethylimino linkedoligonucleotides, also identified as MMI linked oligonucleotides,methylenedimethyl-hydrazo linked oligonucleotides, also identified asMDII linked oligonucleotides, and methylenecarbonylamino linkedoligonucleotides, also identified as amide-3 linked oligonucleotides,and methyleneaminocarbonyl linked oligo-nucleotides, also identified asamide-4 linked oligonucleo-sides, as well as mixed backbone compoundshaving, for instance, alternating MMI and P═O or P═S linkages can beprepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677,5,602,240 and 5,610,289; formacetal and thioformacetal linkedoligonucleotides can be prepared as described in U.S. Pat. Nos.5,264,562 and 5,264,564; and ethylene oxide linked oligonucleotides canbe prepared as described in U.S. Pat. No. 5,223,618. Each of the citedpatents and patent applications is incorporated by reference herein inits entirety.

[0277] Oligonucleotide Formulations and Pharmaceutical Compositions

[0278] The present oligonucleotides can be prepared in anoligonucleotide formulation or pharmaceutical composition. Thus, thepresent oligonucleotides may also be admixed, encapsulated, conjugatedor otherwise associated with other molecules, molecule structures ormixtures of compounds, as for example, liposomes, receptor targetedmolecules, oral, rectal, topical or other formulations, for assisting inuptake, distribution and/or absorption. Exemplary United States patentsthat describe the preparation of such uptake, distribution and/orabsorption assisting formulations include, for example, U.S. Pat. Nos.5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158;5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556;5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619;5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528;5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of whichis incorporated herein by reference in its entirety.

[0279] The oligonucleotides, formulations, and compositions of theinvention include any pharmaceutically acceptable salts, esters, orsalts of such esters, or any other compound which, upon administrationto an animal including a human, is capable of providing (directly orindirectly) the biologically active metabolite or residue thereof.Accordingly, for example, the disclosure is also drawn to prodrugs andpharmaceutically acceptable salts of the compounds of the invention,pharmaceutically acceptable salts of such prodrugs, and otherbioequivalents.

[0280] The term “prodrug” indicates a therapeutic agent that is preparedin an inactive form that is converted to an active form (i.e., drug)within the body or cells thereof by the action of endogenous enzymes orother chemicals and/or conditions. In particular embodiments, prodrugversions of the present oligonucleotides are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in Gosselin et al., WO 93/24510 and in Imbach et al., WO94/26764 and U.S. Pat. No. 5,770,713, which are hereby incorporated byreference in their entireties.

[0281] The term “pharmaceutically acceptable salts” refers tophysiologically and pharmaceutically acceptable salts of the presentcompounds: i.e., salts that retain the desired biological activity ofthe parent compound and do not impart undesired toxicological effectsthereto. Many such pharmaceutically acceptable salts are known and canbe used in the present invention.

[0282] For oligonucleotides, useful examples of pharmaceuticallyacceptable salts include but are not limited to salts formed withcations such as sodium, potassium, ammonium, magnesium, calcium,polyamines such as spermine and spermidine, etc.; acid addition saltsformed with inorganic acids, for example hydrochloric acid, hydrobromicacid, sulfuric acid, phosphoric acid, nitric acid and the like; saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; andsalts formed from elemental anions such as chlorine, bromine, andiodine.

[0283] The present invention also includes pharmaceutical compositionsand formulations which contain the antiviral oligonucleotides of theinvention. Such pharmaceutical compositions may be administered in anumber of ways depending upon whether local or systemic treatment isdesired and upon the area to be treated. For example, administration maybe topical (including ophthalmic and to mucous membranes includingvaginal and rectal delivery); pulmonary, e.g., by inhalation orinsufflation of powders or aerosols, including by nebulizer;intratracheal; intranasal; epidermal and transdermal; oral; orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration.

[0284] Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful. Preferred topical formulationsinclude those in which the oligonucleotides of the invention are inadmixture with a topical delivery agent such as lipids, liposomes, fattyacids, fatty acid esters, steroids, chelating agents and surfactants.Preferred lipids and liposomes include neutral (e.g.dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl cholineDMPC, distearolyphosphatidyl choline) negative (e.g.dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). Oligonucleotides may be encapsulated withinliposomes or may form complexes thereto, in particular to cationicliposomes. Alternatively, oligonucleotides may be complexed to lipids,in particular to cationic lipids. Preferred fatty acids and estersinclude but are not limited arachidonic acid, oleic acid, eicosanoicacid, laurie acid, caprylic acid, capric acid, myristic acid, palmiticacid, stearic acid, linoleic acid, linolenic acid, dicaprate,tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof.

[0285] Compositions and formulations for oral administration includepowders or granules, microparticulates, nanoparticulates, suspensions orsolutions in water or non-aqueous media, capsules, gel capsules,sachets, tablets or minitablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable. Preferred oralformulations are those in which oligonucleotides of the invention areadministered in conjunction with one or more penetration enhancerssurfactants and chelators. Exemplary surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Exemplary bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenedeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate. Exemplaryfatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g. sodium). Also preferred are combinations of penetrationenhancers, for example, fatty acids/salts in combination with bileacids/salts. A particularly preferred combination is the sodium salt oflauric acid, capric acid and UDCA. Further exemplary penetrationenhancers include polyoxyethylene-9-lauryl ether,polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may bedelivered orally in granular form including sprayed dried particles, orcomplexed to form micro or nanoparticles. Oligonucleotide complexingagents include poly-amino acids; polyimines; polyacrytates;polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationizedgelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) andstarches; polyalkylcyanoacrylates; DEAE-derivatized polyimines,pollulans, celluloses, and starches. Particularly advantageouscomplexing agents include chitosan, N-trimethytchitosan, poly-L-lysine,polyhistidine, polyorithine, polyspermines, protamine,polyvinylpyridine, polythiodiethylamino-methylethylene P(TDAE),polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate),poly(ethylcyanoacrylate), poly(butylcyanoacrylatc),poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate),DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin andDEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lacticacid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, andpolyethyleneglycol (PEG).

[0286] Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

[0287] Pharmaceutical compositions of the present invention include, butare not limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

[0288] The pharmaceutical formulations of the present invention, whichmay conveniently be presented in unit dosage form, may be preparedaccording to conventional techniques well known in the pharmaceuticalindustry. Such techniques include the step of bringing into associationthe active ingredients with the pharmaceutical carrier(s) orexcipient(s). In general the formulations are prepared by uniformly andintimately bringing into association the active ingredients with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaking the product.

[0289] The compositions of the present invention may be formulated intoany of many possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

[0290] In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

[0291] Emulsions

[0292] The formulations and compositions of the present invention may beprepared and formulated as emulsions. Emulsions are typicallyheterogenous systems of one liquid dispersed in another in the form ofdroplets usually exceeding 0.1 μm in diameter. (Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (lids.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p.199; Rosoff, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 2, p. 335; Higuchi et at., in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.301). Emulsions are often biphasic systems comprising of two immiscibleliquid phases intimately mixed and dispersed with each other. Ingeneral, emulsions may be either water-in-oil (w/o) or of theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions may contain additional componentsin addition to the dispersed phases and the active drug which may bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants may also be present in emulsions asneeded. Pharmaceutical emulsions may also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous provides an o/w/o emulsion.

[0293] Emulsions are characterized by little or no thermodynamicstability. Often, the dispersed or discontinuous phase of the emulsionis well dispersed into the external or continuous phase and maintainedin this form through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

[0294] Synthetic surfactants, also known as surface active agents, havefound wide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p.199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: non-ionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

[0295] Naturally occurring emulsifiers used in emulsion formulationsinclude lanolin, beeswax, phosphatides, lecithin and acacia. Absorptionbases possess hydrophilic properties such that they can soak up water toform w/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

[0296] A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

[0297] Hydrophilic colloids or hydrocolloids include naturally occurringgums and synthetic polymers such as polysaccharides (for example,acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, andtragacanth), cellulose derivatives (for example, carboxymethylcelluloseand carboxypropylcellulose), and synthetic polymers (for example,carbomers, cellulose ethers, and carboxyvinyl polymers). These disperseor swell in water to form colloidal solutions that stabilize emulsionsby forming strong inter-facial films around the dispersed-phase dropletsand by increasing the viscosity of the external phase.

[0298] Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid, Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

[0299] The application of emulsion formulations via dermatological, oraland parenteral routes and methods for their manufacture have beenreviewed in the literature (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199). Emulsion formulations for oral deliveryhave been very widely used because of reasons of ease of formulation,efficacy from an absorption and bioavailabiity standpoint. (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

[0300] In one embodiment of the present invention, the compositions ofoligonucleotides are formulated as microemulsions. A microemulsion maybe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution(Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).Typically micro-emulsions are systems that are prepared by firstdispersing an oil in an aqueous surfactant solution and then adding asufficient amount of a fourth component, generally an intermediatechain-length alcohol to form a transparent system. Therefore,microemulsions have also been described as thermodynamically stable,isotropically clear dispersions of two immiscible liquids that arestabilized by interfacial films of surface-active molecules (Leung andShah, in: Controlled Release of Drugs: Polymers and Aggregate Systems,Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215).Microemulsions commonly are prepared via a combination of three to fivecomponents that include oil, water, surfactant, cosurfactant andelectrolyte. Whether the microemulsion is of the water-in-oil (w/o) oran oil-in-water (o/w) type is dependent on the properties of the oil andsurfactant used and on the structure and geometric packing of the polarheads and hydrocarbon tails of the surfactant molecules (Schott, inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,1985, p. 271).

[0301] The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

[0302] Surfactants used in the preparation of microemulsions include,but are not limited to, ionic surfactants, non-ionic surfactants, Brij96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (S0750), decaglycerol decaoleate (DA0750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

[0303] Microemulsions are particularly of interest from the standpointof drug solubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994,11,1385-1390; Ritschet, Met/i. Find. Exp.Clin. PharmacoL, 1993, 13, 205). Micro-emulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset at., Pharmaceutical Research, 1994,11, 1385; Ho et al., J. Pharm.Set, 1996, 85,138-143). Often microemulsions may form spontaneously whentheir components are brought together at ambient temperature. This maybe particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucteotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

[0304] Microemulsions of the present invention may also containadditional components and additives such as sorbitan monostearate (Grill3), Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories-surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92).

[0305] Liposomes

[0306] There are many organized surfactant structures besidesmicroemulsions that have been studied and used for the formulation ofdrugs. These include monolayers, micelles, bilayers and vesicles.Vesicles offer specificity and extended duration of action for drugdelivery. Thus, as used herein, the term “liposome” refers to a vesiclecomposed of amphiphilic lipids arranged in a spherical bilayer orbilayers, i.e., liposomes are unilamellar or multilamellar vesicleswhich have a membrane formed from a lipophilic material and an aqueousinterior. The aqueous portion typically contains the composition to bedelivered. In order to cross intact mammalian skin, lipid vesicles mustpass through a series of fine pores, each with a diameter less than 50nm, under the influence of a suitable transdermal gradient. Therefore,it is desirable to use a liposome which is highly deformable and able topass through such fine pores. Additional factors for liposomes includethe lipid surface charge, and the aqueous volume of the liposomes.

[0307] Further advantages of liposomes include; liposomes obtained fromnatural phospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245).

[0308] For topical administration, there is evidence that liposomespresent several advantages over other formulations. Such advantagesinclude reduced side-effects related to high systemic absorption of theadministered drug, increased accumulation of the administered drug atthe desired target, and the ability to administer a wide variety ofdrugs, both hydrophilic and hydrophobic, into the skin. Compoundsincluding analgesics, antibodies, hormones and high-molecular weightDNAs have been administered to the skin, generally resulting intargeting of the upper epidermis.

[0309] Liposomes fall into two broad classes. Cationic liposomes arepositively charged liposomes which interact with the negatively chargedDNA molecules to form a stable complex. The positively chargedDNA/liposome complex binds to the negatively charged cell surface and isinternalized in an endosome. Due to the acidic pH within the endosome,the liposomes are ruptured, releasing their contents into the cellcytoplasm (Wang et at., Biochem. Biophys. Res. Commun.,1987,147,980-985).

[0310] Liposomes which are pH-sensitive or negatively-charged, entrapDNA rather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs. TheDNA is thus entrapped in the aqueous interior of these liposomes.pH-sensitive liposomes have been used, for example, to deliver DNAencoding the thymidine kinase gene to cell monolayers in culture (Zhouet al., Journal of Controlled Release, 1992, 19, 269-274).

[0311] One major type of liposomal composition includes phospholipidsother than naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0312] Several studies have assessed the topical delivery of liposomaldrug formulations to the skin. Application of liposomes containinginterferon to guinea pig skin resulted in a reduction of skin herpessores while delivery of interferon via other means (e.g. as a solutionor as an emulsion) were ineffective (Weiner et at., Journal of DrugTargeting, 1992, 2, 405-410). Further, an additional study tested theefficacy of interferon administered as part of a liposomal formulationto the administration of interferon using an aqueous system, andconcluded that the liposomal formulation was superior to aqueousadministration (du Plessis et al., Antiviral Research, 1992,18,259-265).

[0313] Non-ionic liposomal systems have also been examined to determinetheir utility in the delivery of drugs to the skin, in particularsystems comprising non-ionic surfactant and cholesterol. Non-ionicliposomal formulations comprising Novasone™I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et at. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

[0314] Liposomes also include “sterically stabilized” liposomes, a termwhich, as used herein, refers to liposomes comprising one or morespecialized lipids that, when incorporated into liposomes, result inenhanced circulation lifetimes relative to liposomes lacking suchspecialized lipids. Examples of sterically stabilized liposomes arethose in which part of the vesicle-forming lipid portion of the liposomeinclude one or more glycolipids, such as monosialoganglioside G_(MI), oris derivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. Without being bound by any particulartheory, it is believed that for sterically stabilized liposomescontaining gangliosides, sphingomyelin, or PEG-derivatized lipids, theincrease in circulation half-life of these sterically stabilizedliposomes is due to a reduced uptake into cells of thereticuloendothelial system (RES) (Allen et at., FEBS Lett., 1987, 223,42; Wu et al., Cancer Research, 1993, 53, 3765).

[0315] Various liposomes that include one or more glycolipids have beenreported in Papahadjopoulos et al., Ann. N.Y. Acad. Sci., 1987, 507, 64(monosiatoganglioside G_(M1), galactocerebroside sulfate andphosphatidylinositol); Gabizon et at., Proc. Natl. Acad. Sci. USA.,1988, 85, 6949,;Allen et al., U.S. Pat. No. 4,837,028 and InternationalApplication Publication WO 88/04924 (sphingomyelin and the gangliosideG_(M1) or a galactocerebroside sulfate ester); Webb et al., U.S. Pat.No. 5,543,152 (sphingomyelin); Lim et al., WO 97/13499(1,2-sn-dimyristoylphosphatidylcholine).

[0316] Liposomes that include lipids derivatized with one or morehydrophilic polymers, and methods of preparation are described, forexample, in Sunamoto et al., Bull. Chem. Soc. Jpn., 1980, 53, 2778 (anonionic detergent, 2C₁₂15G, that contains a PEG moiety); Illum et al.,FEBS Lett., 1984,167, 79 (hydrophilic coating of polystyrene particleswith polymeric glycols); Sears, U.S. Pat. Nos. 4,426,330 and 4,534,899(synthetic phospholipids modified by the attachment of carboxylic groupsof polyalkylene glycols (e.g., PEG)); Klibanov et al., FEBS Lett., 1990,268, 235 (phosphatidylethanolamine (PE) derivatized with PEG or PEGstearate); Blume et al., Biochimica et Biophysica Acta, 1990, 1029, 91(PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from thecombination of distearoylphosphatidylethanolamine (DSPE) and PEG);Fisher, European Patent No. EP 0 445 131 B1 and WO 90/04384 (covalentlybound PEG moieties on liposome external surface); Woodle et al., U.S.Pat. Nos. 5,013,556 and 5,356,633, and Martin et al., U.S. Pat. No.5,213,804 and European Patent No. EP 0 496 813 B1 (liposome compositionscontaining 1-20 mole percent of PE derivatized with PEG); Martin et al.,WO 91/05545 and U.S. Pat. No. 5,225,212 and in Zalipsky et al., WO94/20073 (liposomes containing a number of other lipid-polymerconjugates); Choi et al., WO 96/10391 (liposomes that includePEG-modified ceramide lipids); Miyazaki et al., U.S. Pat. No. 5,540,935,and Tagawa et al., U.S. Pat. No. 5,556,948 (PEG-containing liposomesthat can be further derivatized with functional moieties on theirsurfaces).

[0317] Liposomes that include nucleic acids have been described, forexample, in Thierry et al., WO 96/40062 (methods for encapsulating highmolecular weight nucleic acids in liposomes); Tagawa et al., U.S. Pat.No. 5,264,221 (protein-bonded liposomes containing RNA); Rahman et al.,U.S. Pat. No. 5,665,710 (methods of encapsulating oligodeoxynucleotidesin liposomes); Love et al., WO 97/04787 (liposomes that includeantisense oligonucleotides).

[0318] Another type of liposome, transfersomes are highly deformablelipid aggregates which are attractive for drug delivery vehicles. (Cevcet al., 1998, Biochim Biophys Acta. 1368(2):201-15.) Transfersomes maybe described as lipid droplets which are so highly deformable that theycan penetrate through pores which are smaller than the droplet.Transfersomes are adaptable to the environment in which they are used,for example, they are shape adaptive, self-repairing, frequently reachtheir targets without fragmenting, and often self-loading. Transfersomescan be made, for example, by adding surface edge-activators, usuallysurfactants, to a standard liposomal composition.

[0319] Surfactants

[0320] Surfactants are widely used in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

[0321] If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants are widely used inpharmaceutical and cosmetic products and are usable over a wide range ofpH values, and with typical HLB values from 2 to about 18 depending onstructure. Nonionic surfactants include nonionic esters such as ethyleneglycol esters, propylene glycol esters, glyceryl esters, polyglycerylesters, sorbitan esters, sucrose esters, and ethoxylated esters; andnonionic alkanolamides and ethers such as fatty alcohol ethoxylates,propoxylated alcohols, and ethoxylated/propoxylated block polymers arealso included in this class. The polyoxyethylene surfactants are themost commonly used members of the nonionic surfactant class.

[0322] Surfactant molecules that carry a negative charge when dissolvedor dispersed in water are classified as anionic. Anionic surfactantsinclude carboxylates such as soaps, acyl lactylates, acyl amides ofamino acids, esters of sulfuric acid such as alkyl sulfates andethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates,acyl isothionates, acyl laurates and sulfosuccinates, and phosphates.The alkyl sulfates and soaps are the most commonly used anionicsurfactants.

[0323] Surfactant molecules that carry a positive charge when dissolvedor dispersed in water are classified as cationic. Cationic surfactantsinclude quaternary ammonium salts and ethoxylated amines, with thequaternary ammonium salts used most often.

[0324] Surfactant molecules that can carry either a positive or negativecharge are classified as amphoteric. Amphoteric surfactants includeacrylic acid derivatives, substituted alkylamides, N-alkylbetaines andphosphatides.

[0325] The use of surfactants in drug products, formulations and inemulsions has been reviewed in Rieger, in Pharmaceutical Dosage Forms,Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0326] Penetration Enhancers

[0327] In some embodiments, penetration enhancers are used in or with acomposition to increase the delivery of nucleic acids, particularlyoligonucleotides, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs may cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

[0328] Penetration enhancers may be classified as belonging to one offive broad categories, i.e., surfactants, fatty acids, bile salts,chelating agents, and non-chelating nonsurfactants (Lee et al., CriticalReviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of theseclasses of penetration enhancers is described below in greater detail.

[0329] Surfactants: In connection with the present invention,surfactants (or “surface-active agents”) are chemical entities which,when dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of oligonucleotidesthrough the mucosa is enhanced. These penetration enhancers include, forexample, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether andpolyoxyethylene-20-cetyl ether) (Lee et at., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemicalemulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988,40, 252), each of which is incorporated herein by reference in itsentirety.

[0330] Fatty acids: Various fatty acids and their derivatives which actas penetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and diglycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654),each of which is incorporated herein by reference in its entirety.

[0331] Bile salts: The physiological role of bile includes thefacilitation of dispersion and absorption of lipids and fat-solublevitamins (Brunton, Chapter 38 in: Goodman & Gilman's The PharmacologicalBasis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, NewYork, 1996, pp. 934-935). Various natural bile salts, and theirsynthetic derivatives, act as penetration enhancers. Thus the term “bilesalts” includes any of the naturally occurring components of bile aswell as any of their synthetic derivatives. The bile salts of theinvention include, for example, cholic acid (or its pharmaceuticallyacceptable sodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1-33; Yamamoto ct al., J. Pharm. Exp. Ther., 1992,263, 25; Yamashita et al., J. Pharm:. Sci., 1990, 79, 579-583).

[0332] Chelating Agents: In the present context, chelating agents can beregarded as compounds that remove metallic ions from solution by formingcomplexes therewith, with the result that absorption of oligonucleotidesthrough the mucosa is enhanced. With regards to their use as penetrationenhancers in the present invention, chelating agents have the addedadvantage of also serving as DNase inhibitors, as most characterized DNAnucleases require a divalent metal ion for catalysis and are thusinhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618,315-339). Without limitation, chelating agents include disodiumethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g.,sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines)(Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7,1-33; Buur et al., J. ControlRel., 1990, 14, 43-51).

[0333] Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds are compounds that do notdemonstrate significant chelating agent or surfactant activity, butstill enhance absorption of oligonucleotides through the alimentarymucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33). Examples of such penetration enhancers includeunsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanonederivatives (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, page 92); and nonsteroidal anti-inflammatory agents suchas diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al,J. Pharm. Pharmacol., 1987, 39, 621-626).

[0334] Agents that enhance uptake of oligonucleotides at the cellularlevel may also be added to the pharmaceutical and other compositions andformulations of the present invention. For example, cationic lipids,such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationicglycerol derivatives, and polycationic molecules, such as polylysine(Lollo et al., PCT Application WO 97/30731), are also known to enhancethe cellular uptake of oligonucleotides.

[0335] Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

[0336] Carriers

[0337] Certain compositions of the present invention also incorporatecarrier compounds in the formulation. As used herein, “carrier compound”or “carrier” can refer to a nucleic acid, or analog thereof, which isinert (i.e., does not possess biological activity per se) but isrecognized as a nucleic acid by in vivo processes that reduce thebioavailability of a nucleic acid having biological activity by, forexample, degrading the biologically active nucleic acid or promoting itsremoval from circulation. The coadministration of a nucleic acid and acarrier compound, often with an excess of the latter substance, canresult in a substantial reduction of the amount of nucleic acidrecovered in the liver, kidney or other extracirculatory reservoirs. Forexample, the recovery of a partially phosphorothioate oligonucleotide inhepatic tissue can be reduced when it is coadministered withpolyinosinic acid, dextran sulfate, polycytidic acid or4-acetamido-4′isothiocyano-stilbene-2,2-disulfonic acid (Miyao et al.,AntisenseRes. Dev., 1995,5,115-121; Takakura et al., Antisense & Nucl.Acid Drug Dev., 1996, 6, 177-183), each of which is incorporated hereinby reference in its entirety.

[0338] Excipients

[0339] In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal, and is typically liquid or solid. Apharmaceutical carrier is generally selected to provide for the desiredbulk, consistency, etc., when combined with a nucleic acid and the othercomponents of a given pharmaceutical composition, in view of theintended administration mode. Typical pharmaceutical carriers include,but are not limited to, binding agents (e.g., pregelatinized maizestarch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.);fillers (e.g., lactose and other sugars, microcrystalline cellulose,pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates orcalcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate,talc, silica, colloidal silicon dioxide, stearic acid, metallicstearates, hydrogenated vegetable oils, corn starch, polyethyleneglycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g.,starch, sodium starch glycotate, etc.); and wetting agents (e.g., sodiumlauryl sulphate, etc.).

[0340] Pharmaceutically acceptable organic or inorganic excipientssuitable for non-parenteral administration which do not deleteriouslyreact with nucleic acids can also be used to formulate the compositionsof the present invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

[0341] Formulations for topical administration of nucleic acids mayinclude sterile and non-sterile aqueous solutions, non-aqueous solutionsin common solvents such as alcohols, or solutions of the nucleic acidsin liquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

[0342] Other Pharmaceutical Composition Components

[0343] The present compositions may additionally contain othercomponents conventionally found in pharmaceutical compositions, at theirart-established usage levels. Thus, for example, the compositions maycontain additional, compatible, pharmaceutically-active materials suchas, for example, antipruritics, astringents, local anesthetics oranti-inflammatory agents, or may contain additional materials useful inphysically formulating various dosage forms of the compositions of thepresent invention, such as dyes, flavoring agents, preservatives,antioxidants, opacifiers, thickening agents and stabilizers. However,such materials, when added, should not unduly interfere with thebiological activities of the components of the compositions of thepresent invention. The formulations can be sterilized and, if desired,mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, colorings, flavorings and/or aromatic substances andthe like which do not deleteriously interact with the nucleic acid(s) ofthe formulation.

[0344] Aqueous suspensions may contain substances which increase theviscosity of the suspension including, for example, sodiumcarboxymethylcellulose, sorbitol and/or dextran, and/or stabilizers.

[0345] Certain embodiments of the invention provide pharmaceuticalcompositions containing (a) one or more antiviral oligonucleotides and(b) one or more other chemotherapeutic agents which function by adifferent mechanism. Examples of such chemotherapeutic agents includebut are not limited to daunorubicin, daunomycin, dactinomycin,doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide,ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan,mitomycin C, actinomycin D, mithramycin, prednisone,hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine,hexamethylmelamine, pentamethytmetamine, mitoxantrone, amsacrine,chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin, and diethylstilbestrol(DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15thEd. 1987, pp.1206-1228, Berkow et al., eds., Rahway, N.J. When used withthe compounds of the invention, such chemotherapeutic agents may be usedindividually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FUand oligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-EU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide). Anti-inflammatory drugs, includingbut not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, and antiviral drugs, including but not limited toRibavirin, cidofovir, vidarabine, acyclovir and ganciclovir, may also becombined in compositions of the invention. See, generally, The MerckManual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987,Rahway, N.J., pages 2499-2506 and 46-49, respectively). Othernon-oligonucleotide chemotherapeutic agents are also within the scope ofthis invention. Two or more combined compounds may be used together orsequentially.

EXAMPLES Example 1 Herpes Simplex Virus

[0346] Herpes simplex virus (HSV) affects a significant proportion ofthe human population. It was found in the present invention that randomODNs or ODN randomers inhibited the infectivity of viruses such as HSV.Using cellular HSV replication assays in VERO cells (susceptible toHSV-1 (strain KOS) and HSV-2 (strain MS2) infection) it was found that asingle stranded PS-ODN complementary to the HSV origin of replicationinhibited replication of HSV-1 and HSV-2. Surprisingly, control PS-ODNscomplementary to human (343 ARS) and plasmid (pBR322/pUC) origins alsoinhibited viral infectivity. Experiments with random sequence PS-ODNsand PS-ODN randomers demonstrated that inhibition of viral infectionincreased with increasing ODN size. These data show that ONs are potentantiviral agents useful for therapeutic treatment of viral infection.

[0347] The inventors have theorized that a potential mechanism forblocking the spread of viruses such as HHVs was to prevent thereplication of its DNA. With this in mind, phosphorothioateoligonucleotides (ODNs) complementary to the origin of replication ofHSV1 and HSV2 were introduced into infected cells. These ODNs wouldcause DNA triplex formation at the viral origin of replication, blockingthe association of necessary trans-acting factors and viral DNAreplication. Surprising results are presented herein of theseexperiments which show that, in an experimental paradigm, the potency ofODNs in inhibiting viral infection increases as their size (length)increases.

[0348] Inhibition of HSV-1

[0349] The ability of PS-ODNs to inhibit HSV-1 is measured in a plaquereduction assay (PRA). Immortalized African Green Monkey kidney (VERO)cells are cultured at 37° C. and 5% CO₂ in MEM (minimal essentialmedium) plus 10% fetal calf serum supplemented with gentamycin,vancomycin and amphoterecin B. Cells are seeded in 12 well plates at adensity which yields a confluent monolayer of cells after 4 days ofgrowth. Upon reaching confluency, the media is changed to contain only5% serum plus supplements as described above and cells are then exposedto HSV-1 (strain KOS, approximately 40-60 PFU total) in the presence ofthe test compound for 90 minutes. After viral exposure, the media isreplaced with new “overlay” media containing 5% serum, 1% humanimmunoglobulins, supplements as described above and the test compound.Plaque counting is performed 3-4 days post infection following formalinfixation and cresyl violet staining of infected cultures.

[0350] All ONs (except where noted otherwise) were synthesized at theUniversity of Calgary Core DNA Services lab. ONs (see table 1) areprepared on a 1 or 15 micromol synthesis scale, deprotected and desaltedon a 50 cm Sephadex G-25 column. The resulting ONs are analyzed by UVshadowing gel electrophoresis and are determined to contain ˜95% of thefull length, n-1 and n-2 oligo and up to 5% of shorter oligo species(these are assumed to have random deletions). For random oligosynthesis, adenine, guanosine, cytosine and thymidine amidites are mixedtogether in equimolar quantities to maximize the randomness ofincorporation at each position of the ODNs during synthesis.

[0351] To test if PS-ODNs could inhibit HSV-1, REP 1001, 2001 and 3007are tested in the HSV-1 PRA. It is expected that only REP 2001 will showany activity as this PS-ODN is directed against the origin ofreplication in HSV (the other two are directed against replicationorigins in humans and plasmids). However all three PS-ODNs showedanti-HSV-1 activity (see FIG. 1). Moreover, the potentcy of theanti-HSV-1 effect is dependent on the size of the oligo (see FIG. 2).

[0352] To confirm the size dependence and relative sequence independenceof PS-ODNs on anti-HSV-1 activity, we tested PS-ODNs that vary in size(REP 2002, 2003, 2004, 2005 and 2006). These PS-ODNs are rendered inertwith respect to sequence specific effects by synthesizing each base as a“wobble” (N) so that each PS-ODN actually represents a population ofdifferent random sequences with the same size, these PS-ODNs are termed“randomers”. When these oligos are tested in the HSV-1 PRA, we find thatoligos 10 bases or lower have no detectable anti-HSV-1 activity but asthe size of the PS-ODN increases above 10 bases, the potency alsoincreases (IC₅₀ decreases, see FIGS. 3 and 4). We also note that PS-ODNsgreater than 20 bases had IC₅₀ values significantly lower than aclinically accepted anti-HSV-1 drug, acyclovir (see FIG. 4).

[0353] To better define the effective size range for PS-ODN anti-HSV-1activity, we tested PS-ODN randomers covering a broader range of sizesfrom 10 to 120 bases (see FIGS. 5 and 6). We discovered that oligos 12bases and larger have detectable anti-HSV-1 activity and that theefficacy against HSV-1 also increases with increased PS-ODN randomerlength at least up to 120 bases. However, the increases in efficacy perbase increase in size are smaller in PS-ODN randomers greater than 40bases (see FIG. 7).

[0354] To compare the efficacy of non-PS-ODN randomers, a randomsequence PS-ODN and a HSV-1 specific sequence PS-ODN, we tested thesethree types of modifications in ODNs 10, 20 and 40 bases in size (seeFIGS. 8 and 9). Unmodified ODN randomers have no detectable anti-HSV-1activity at tested sizes (see FIGS. 8a-c). Both random sequence andspecific HSV-1 sequence PS-ODNs show size dependent anti-HSV-1 activity(no activity is observed at 10 bases for either of these modifications,see FIGS. 8d and g). A comparison of random sequence, specific HSV-1sequence and randomer PS-ODNs (see FIG. 10) shows that for PS-ODNs 20bases in length, there is an enhancement of anti-HSV-1 activity with thespecific HSV-1 sequence but that at 40 bases in length, allmodifications, whether randomer, random sequence or specific HSV-1sequence were equally efficacious against HSV-1.

[0355] To the best of our knowledge, this is the first time IC50s forHSV-1 as low as 0.059 μM and 0.043 μM are reported for PS-ODNs.

EXAMPLE 2 Inhibition of HSV-2

[0356] The ability of PS-ODNs to inhibit HSV-2 is measured by PRA.Immortalized African Green Monkey kidney (VERO) cells are cultured at37° C. and 5% CO₂ in MEM plus 10% fetal calf serum supplemented withgentamycin, vancomycin and amphoterecin B. Cells are seeded in 12 wellplates at a density which yields a confluent monolayer of cells after 4days of growth. Upon reaching confluency, the media is changed tocontain only 5% serum plus supplements as described above and cells arethen exposed to HSV-2 (strain MS2, approximately 40-60 PFU total) in thepresence of the test compound for 90 minutes. After viral exposure, themedia is replaced with new “overlay” media containing 5% serum, 1% humanimmunoglobulins, supplements as described above and the test compound.Plaque counting is performed 3-4 days post infection following formalinfixation and cresyl violet staining of infected cultures.

[0357] To test if PS-ODNs could inhibit HSV-2, REP 1001, 2001 and 3007are tested in the HSV-2 PRA. It is expected that only REP 2001 will showany activity as this PS-ODN is directed against the origin ofreplication in HSV-1/2 (the other two are directed against replicationorigins in humans and plasmids), however all three PS-ODNs showedanti-HSV-2 activity (see FIG. 12). Moreover, the potency of theanti-HSV-2 effect is dependent on the size of the PS-ODN and independentof the sequence (see FIG. 13).

[0358] To confirm the size dependence and sequence independence ofPS-ODNs on anti-HSV-2 activity, we test PS-ODNs that vary in size (REP2001, 2002, 2003, 2004, 2005 and 2006). These PS-ODNs are rendered inertwith respect to sequence specific effects by synthesizing each base as a“wobble” (N) so that each PS-ODN actually represents a population ofdifferent random sequences with the same size, these PS-ODNs are termed“randomers”. When these PS-ODNs are tested in the HSV-2 PRA, we findthat PS-ODNs 10 bases or lower had no detectable anti-HSV-2 activity butas the size of the PS-ODN increases above 10 bases, the potency alsoincreases (IC₅₀ decreases, see FIGS. 14 and 15). We also noted thatPS-ODNs greater than 20 bases had IC₅₀ values significantly lower than aclinically accepted anti-HSV-2 drug, acyclovir™ (see FIG. 15).

[0359] To the best of our knowledge, this is the first time an IC50 forHSV-2 as low as 0.012 μ/M has been reported for a PS-ODN.

[0360] To determine if non-specific sequence composition has an effecton ON antiviral activity, several PS-ODNs of equivalent size butdiffering in their sequence composition were tested for anti-HSV1activity in the HSV-1 PRA. The PS-ODNs tested were REP 2006 (N20), REP2028 (G40), REP 2029 (A40), REP 2030 (T40) and REP 2031 (C40). The IC50values generated from the HSV-1 PRA (see FIG. 37) show that REP 2006(N40) was clearly the most active of all sequences tested while REP 2029(A40) was the least active. We also note that, all the other PS-ODNswere significantly less active than N40 with their rank in terms ofefficacy being N40>C40>T40>A40>>G40.

[0361] We also tested the efficacy of different PS ODNs having varyingsequence composition with two different nucleotides (see FIG. 37b). ThePS-ODN randomer (REP 2006) was significantly more efficacious againstHSV-1 than AC20 (REP 2055), TC20 (REP 2056) or AG20 (REP 2057) withtheir efficacies ranked as follows: N40>AG>AC>TC. This data suggeststhat although the anti-viral effect is non-sequence complementary,certain non-specific sequence compositions (ie C40 and N40) have themost potent anti-viral activity. We suggest that this phenomenon can beexplained by the fact that, while retaining intrinsic protein bindingability, sequences like C40, A40, T40 and G40 bind fewer viral proteinswith high affinity, probably due to some restrictive tertiary structureformed in these sequences. On the other hand, due to the random natureof N40, it retains its ability to bind with high affinity to a broadrange of anti-viral proteins which contributes to its robust anti-viralactivity.

EXAMPLE 3 Inhibition of CMV

[0362] The ability of PS-ODNs to inhibit CMV is measured in a plaquereduction assay (PRA). This assay is identical to the assay used fortesting anti-HSV-1 and anti-HSV-2 except that CMV (strain AD169) is usedas the viral innoculum and human fibroblasts were used as cellular host.

[0363] To test the size dependence and sequence independence of PS-ODNson anti-CMV activity, we test PS-ODN randomers that vary in size (seeFIGS. 16a, b). When these PS-ODNs are tested in the CMV PRA, we findthat as the size of the PS-ODN increases, the potency also increases(IC₅₀ decreases, see FIG. 16c).

[0364] To more clearly elucidate the effective size range for PS-ODNanti-CMV activity, we tested PS-ODN randomers covering a broader rangeof sizes from 10 to 80 bases. We also included several clinicallyaccepted small molecule CMV treatments (Gancyclovir, Foscarnet andCidofovir) as well as 2 versions of a marketed antisense treatment forCMV retinitis, (Vitravene™; commercially available and synthesized bythe University of Calgary) (see FIG. 17). We discovered that whileincreased PS-ODN randomer size leads to increased efficacy, this effectsaturates at 40 bases (see FIG. 18). Moreover, the 20, 40 and 80 basePS-ODN randomers are all significantly more efficacious than any of thesmall molecule treatments tested (FIG. 17). In addition, 40 and 80 basePS-ODN randomers are more efficacious than Vitravene™.

[0365] To the best of our knowledge, this is the first time an IC50 forCMV as low as 0.067 μM has been reported for a PS-ODN.

EXAMPLE 4 Inhibition of HIV-1

[0366] The ability of PS-ODN randomers to inhibit HIV-1 is measured bytwo different assays:

[0367] Cytopathic Effect (CPE)

[0368] Cytopathic effect is monitored using MTT dye to report the extentof cellular metabolism. Immortalized human lymphocyte (MT4) cells arecultured at 37° C. and 5% CO₂ in MEM plus 10% fetal calf serumsupplemented with antibiotics. Cells are seeded in 96 well plates inmedia containing the appropriate test compound and incubated for 2hours. After preincubation with the test compound, HIV-1 (strain NL 4-3)was added to the wells (0.0002 TCID₅O/cell). After 6 days of additionalincubation, CPE is monitored by MTT conversion. Cytotoxicity is measuredby incubating the drugs for 6 days in the absence of viral inoculation.For transformation of MTT absorbance values into % survival, theabsorbance of uninfected, untreated cells is set to 100% and theabsorbance of infected, untreated cells is set to 0%.

[0369] Replication Assay (RA)

[0370] The ability of HIV to replicate is monitored in immortalizedhuman embryonic kidney (293A) cells. These cells are cotransfected withtwo plasmids. One plasmid contains a recombinant wild type HIV-1 genome(NL 4-3) having its env gene disrupted by a luciferase expressioncassette (identified as strain CNDO), the other plasmid contains the envgene from murine leukemia virus (MLV). These two plasmids provide allthe protein factors in trans to produce a mature chimeric virus havingall the components from HIV-1 except the protein products provided intrans from the MLV env gene. Virions produced from these cells areinfectious and replicative but cannot produce another generation ofinfectious virions because they will lack the env gene products.

[0371] 24 hours after transfection, these cells are trypsinized andplated in 96 well plates. After the cells have adhered, the media iswashed and replaced with media containing the test compound. Virusproduction is allowed to proceed for an additional 24 hours. Thesupernatant is then harvested and used to reinfect naive 293A cells.Naive cells that are infected are identified by the luciferase geneproduct. The number of luciferase positive cells is a measure of theextent of replication and/or infection by the recombinant HIV-1. Thisassay is also adapted to test the resistance to many clinically acceptedanti-HIV-1 drugs by using a HIV-1 genome with several point mutationsknown to induce resistance to several different classes of anti-HIVdrugs. Percentage inhibition is set to 100% for no detectable luciferasepositive cells and 0% for the number of positive cells in infected,untreated controls.

[0372] To test the size dependence and sequence independence of PS-ODNson anti-HIV-1 activity, we test PS-ODN randomers that vary in size. Whenthese PS-ODN randomers are tested the HIV-1 CPE assay, we find that asthe size of the PS-ODN increases the potency also increases (IC₅₀decreases, see FIGS. 19a, b and 20). We also noted that the PS-ODNrandomers exhibited no significant toxicity to the host cells in thisassay (see FIGS. 19c,d).

[0373] To the best of our knowledge, this is the first time an IC50 forHIV-1 as low as 0.011 pM has been reported for a PS-ODN.

[0374] To more clearly elucidate the effective size range for PS-ODNanti-HIV-1 activity, we tested more PS-ODN randomers covering a broaderrange of sizes from 10 to 80 bases by RA using wild-type HIV-1(recombinant NL 4-3 (CNDO)). In addition, we tested four proteaseinhibitors currently used in the clinic (aprenavir, indinavir, lopinavirand saquinavir). We discovered that PS-ODN randomers 10 bases and largerhave anti-HIV-1 activity and that the efficacy against HIV-1 alsoincreases with increased PS-ODN randomer length but is saturated at 40bases (see FIGS. 21e-h and FIG. 22b). Moreover, the 40 and 80 basePS-ODN randomers were almost equivalent in efficacy with the 4 clinicalcontrols (see FIGS. 21a-d and 22 a).

[0375] To the best of our knowledge, this is the first time an IC50 forHIV-1 as low as 0.014 μM has been reported for a PS-ODN.

[0376] To test the ability of PS-ODN randomers to inhibit a drugresistant strain of HIV, we duplicated the above test using therecombinant MDRC4 strain of HIV-1. This recombinant strain exhibitssignificant resistance to at least 16 different clinically accepteddrugs from all classes: nucleotide RT inhibitors, non-nucleotide RTinhibitors and protease inhibitors. All the PS-ODN randomers perform aswell against the resistant strain as they do against the wild typestrain (see FIGS. 23e-h). However, three of the four protease inhibitorsshow a reduction in their efficacy against the mutant strain (see FIGS.23a-d and 24), such that the 40 and 80 base PS-ODN randomers are nowmore potent against this resistant strain than these drugs.

EXAMPLE 5 Inhibition of RSV

[0377] The ability of PS-ODN randomers to inhibit RSV is measured bymonitoring CPE with alamar blue (an indirect measure of cellularmetabolism). Human larynx carcinoma (Hep2) cells are cultured at 37° C.and 5% CO₂ in MEM plus 5% fetal calf serum. Cells are seeded in 96 wellplates at a density which yields a confluent monolayer of cells after5-6 days of growth. The day after plating, cells were infected with RSV(strain A2, 10^(8.2)TCID₅₀/ml) in the presence of the test compound in areduced volume for 2 hours. Following inoculation, the media was changedand was supplemented with test compound. 6 days after infection, CPE wasmonitored by measuring the fluorescent conversion of alamar blue.Toxicity of test compounds in Hep2 cells was monitored by treatinguninfected cells for 7 days and measuring alamar blue conversion inthese cells. The alamar blue readings in uninfected, untreated cellswere set to 100% survival and the readings in infected, untreated cellswere set to 0% survival.

[0378] To confirm the size dependence and sequence independence ofPS-ODNs on anti-RSV activity, we test PS-ODN randomers that vary insize. In addition, we tested the clinically accepted treatment for RSVinfection, Ribavirin (Virazole™). When tested in the RSV CPE assay, wefind that as the size of the PS-ODN randomer increases, the potency alsoincreases but saturates at 40 bases in size (see FIGS. 25a-c and 26). Wealso noted that 20, 40 and 80 base PS-ODN randomers had IC₅₀ valuessignificantly lower than a clinically accepted anti-RSV drug, Ribavirin(see FIGS. 25a-d and 26). PS-ODN randomers exhibited no toxicity in Hep2cells while Ribavirin was significantly toxic (therapeutic index=2.08,see FIGS. 25e-h).

[0379] To the best of our knowledge, this is the first time an IC50 forRSV-1 as low as 0.015 μM has been reported for a PS-ODN.

EXAMPLE 6 Inhibition of Coxsackie virus B2

[0380] The ability of PS-ODN randomers to inhibit COX B2 is measuredmonitoring CPE with alamar blue (an indirect measure of cellularmetabolism). Rhesus monkey kidney (LLC-MK2) cells are cultured at 37° C.and 5% CO₂ in MEM plus 5% fetal calf serum. Cells are seeded in 96 wellplates at a density which yields a confluent monolayer of cells after5-6 days of growth. The day after plating, cells were infected with COXB2 (strain Ohio-1, 107.8 TCID₅₀/ml) in the presence of the test compoundin a reduced volume for 2 hours. Following inoculation, the media waschanged and was supplemented with test compound. 6 days after infection,CPE was monitored by measuring the fluorescent conversion of alamarblue. Toxicity of test compounds in LLC-MK2 cells was monitored bytreating uninfected cells for 7 days and measuring, alamar blueconversion in these cells. The alamar blue readings in uninfected,untreated cells were set to 100% survival and the readings in infected,untreated cells were set to 0% survival.

[0381] We tested the anti-COX B2 activity of REP 2006 in the COX B2 CPEassay. We found that, while exhibiting some slight toxicity in LLC-MK2cells (see FIG. 27b), this PS-ODN randomer was able to partially rescueinfected LLC-MK2 cells from COX B2 infection (see FIG. 27a).

EXAMPLE 7 Inhibition of Vaccinia Virus

[0382] We used the vaccinia infection model as a measure of thepotential efficacy of our compounds against poxviruses, includingsmallpox virus. The ability of PS-ODN randomers to inhibit Vaccinia ismeasured by monitoring CPE with alamar blue (an indirect measure ofcellular metabolism). Vero cells are cultured at 37° C. and 5% CO₂ inMEM plus 5% fetal calf serum. Cells are seeded in 96 well plates at adensity which yields a confluent monolayer of cells after 5-6 days ofgrowth. The day after plating, cells were infected with Vaccinia(10^(7.9) TCID₅O/ml) in the presence of the test compound in a reducedvolume for 2 hours. Following inoculation, the media was changed and wassupplemented with test compound (all at 10 uM, except for Cidofovirwhich was used at 50 uM). Five days after infection, the supernatantswere harvested. The viral load in the supernatant was determined byreinfection of VERO cells with supernatant diluted 1:100 and themonitoring of CPE 7 days after reinfection by measuring the fluorescentconversion of alamar blue.

[0383] We tested PS-ODN randomers that vary in size (REP 2004, 2006 and2007). In addition, we tested a known effective treatment for Vacciniainfection, Cidofovir (Vistide™). When tested in the Vacinnia CPE assay,we find that treatment with REP 2004, 2006 and 2007 all displayedantiviral activity (ie. resulted in supernatants which showed adecreased CPE upon reinfection) but that this activity was weaker thanthat seen for Cidofovir (see FIG. 36).

EXAMPLE 8 Inhibition of DHBV, Parainfluenza-3 Virus, and Hanta Virus

[0384] Because DHBV, Parainfluenza-3 virus and Hanta virus do notreadily generate measurable plaques or CPE, we tested the efficacy ofREP 2006 in these viruses using a fluorescence focus forming unit (FFFU)detection. In this assay, REP 2006 (at a final concentration of 10 uM)is mixed with the virus which is then adsorbed onto the cells. Afteradsorption, infected cells are allowed to incubate for an additional7-14 days at which point they are fixed in methanol. Regions of viralreplication are detected by immunofluorescence microscopy against theappropriate viral antigen. For each of the three viruses tested, thespecific experimental conditions and results are described below: FFFUcount Antibody for FFFU count (10 uM REP Virus Cellular Host FFFUdetection (no drug) 2006) DHBV (HBV Primary duck Mouse anti-DHBV  163+/− 38.5 0 surrogate) hepatocytes IgG Parainfluenza-3 LLC-MK2 Mouseanti-PI3 288 +/− 126 0 cells IgG Hanta Virus VERO E6 Mouse anti- 232.3+/− 38.17 0 (Strain cells SinNombre Prospect Hill) nucleoprotein IgG

[0385] This initial data shows that at 10 uM, REP 2006 is effective ininhibiting DHBV, Parainfluenza-2 and Hanta Virus. We anticipate thatgiven the robust response in the preliminary test that IC₅₀ values willbe lower. These data support the efficacy of PS-ODN randomers for thetreatment of human infections of Hanta Virus and Hepatitis B (closelyrelated to DHBV) as well as providing a rationale for the immediatetreatment of pediatric bronchiolitis caused by RSV and Parainfluenza-3,which may not require differential diagnosis for treatment to begin.

EXAMPLE 9 Currently Non-Responsive Viruses

[0386] To date we have not observed a detectable anti-viral efficacywith PS-ODN randomers (up to 10 uM) without using a delivery system, adrug combination, or a chemical modification in the following viralsystems: Assay Virus Strain Cellular Host paradigm Influenza A H3N2 MDCKcells Plaque reduction Corona virus MHV2 (mouse) NCTC-1496 cells Plaque(SARS MHV-A59 (mouse) DBT cells reduction surrogate) HCoV-OC43 HRT-18cells (human) BVDV (HCV NA BT cells CPE by surrogate) alamar blueRhinovirus HGP HeLa cells CPE by alamar blue Adenovirus Human Ad5 293Acells Plaque reduction

[0387] Under the current testing procedures, we did not demonstrateactivity. However, the lack of antiviral activity may be due to a lowercellular penetration of the PS-ODN under the conditions of the assays.Nonetheless, additional testing is underway to achieve efficaciousresults with these viruses. These viruses may respond to PS-ODN whenusing a delivery system such as a liposomal formulation, in order toincrease its intracellular concentration. Also in a combination withanother antiviral drug, such as described herein, PS-ODNs may exhibit anantiviral efficacy for these viruses. A chemical modification toincrease intracellular concentration may also be useful to renderPS-ODNs active against these viruses.

[0388] Since we have good evidence that the charge characteristics of aPS-ODN are important for the inhibition of viruses from severaldifferent families, we expect that this charge dependent mechanism forthe inhibition of viral activity has the potential to inhibit theactivity of all encapsidating viruses. The corollary to this is that thelack of detected anti-viral efficacy against those viruses listed inExample 9 suggests that the interaction between the PS-ODN and thestructural proteins of these viruses is not strong enough to prevent theinteraction of viral proteins during the replication of these viruses.One way of achieving efficacy against these viruses is to alter thecharge characteristics of the DNA or anti-viral polymer (e.g.,substituting phosphorodithioate for phosphorothioate linkages in DNA) sotheir affinity for viral proteins is increased.

EXAMPLE 10 Tests for Determining if an Oligonucleotide ActsPredominantly by a Non-Sequence Complementary Mode of Action

[0389] An ON, e.g., ODN, in question shall be considered to be actingpredominantly by a non-sequence complementary mode of action if it meetsthe criterion of any one of the 3 tests outlined below.

[0390] Test #1—Effect of Partial Degeneracy on Antiviral Efficacy

[0391] This test serves to measure the antiviral activity of aparticular ON sequence when part of its sequence is made degenerate. Ifthe degenerate version of the ON having the same chemistry retains itsactivity as described below, is it deemed to be acting predominantly bya non-sequence complementary mode of action. ONs will be made degenerateaccording to the following rule:

[0392] L_(ON)=the number of bases in the original ON

[0393] X=the number of bases on each end of the oligo to be madedegenerate (but having the same chemistry as the original ON)

[0394] If L_(ON) is even, then X=L_(ON)/4

[0395] If L_(ON) is odd, then X=integer (L_(ON)/4)+1

[0396] Each degenerate base shall be synthesized according to anysuitable methodology, e.g., the methodology described herein for thesynthesis of PS-ODN randomers.

[0397] If the ON is claimed to have an anti-viral activity against amember of the herpesviridae, retroviridae, or paramyxoviridae families,the IC₅₀ generation will be performed using the assay described hereinfor that viral family preferably using the viral strains indicated. Ifthe ON is claimed to have an anti-viral activity against a member of aparticular virus family not mentioned above, then the IC50 values shallbe generated by a test of antiviral efficacy accepted by thepharmaceutical industry. IC50 values shall be generated using a minimumof seven concentrations of compound, with three or more points in thelinear range of the dose response curve. Using this test, the IC₅₀ ofsaid ON shall be compared to its degenerate counterpart. If the IC₅₀ ofthe degenerate ON is less than 2-fold greater than the original ON foran ON of 25 bases and less, or is less than 10-fold greater than theoriginal ON for ONs 26 bases or more (based on minimum triplicatemeasurements, standard deviation not to exceed 15% of mean) then the ONshall be deemed to be functioning predominantly by a non-sequencecomplementary mode of action.

[0398] Test #2—Comparison of Efficacy With Randomer

[0399] This test serves to compare the anti-viral efficacy of an ON withthe antiviral efficacy of a randomer ON of equivalent size and the samechemistry in the same virus or viral family.

[0400] If the ON is claimed to have an anti-viral activity against amember of the herpesviridae, retroviridae, or paramyxoviridae families,the IC₅₀ generation will be performed using the assay described hereinfor that viral family preferably using the viral strains indicated. Ifthe ON is claimed to have an anti-viral activity against a member of aparticular virus family not mentioned above, then the IC50 values shallbe generated by a test of antiviral efficacy accepted by thepharmaceutical industry. IC50 values shall be generated using a minimumof seven concentrations of compound, with three or more points in thelinear range of the dose response curve. Using this test, the IC₅₀ ofthe ON shall be compared to an ON randomer of equivalent size and thesame chemistry. If the IC₅₀ of the degenerate ON is less than 2-foldgreater than the original ON for an ON of 25 bases and less, or is lessthan 10-fold greater than the original ON for ONs 26 bases or more(based on minimum triplicate measurements, standard deviation not toexceed 15% of mean) then the ON shall be deemed to be functioningpredominantly by a non-sequence complementary mode of action.

[0401] Test #3—Comparison of Efficacy in a Different Viral Family

[0402] This test serves to compare the efficacy of an ON against atarget virus whose genome is homologous to the ON with the efficacy ofthe ON against a second virus whose genome has no homology to ON. Inmany cases, the different virus will be selected from a different viralfamily than the viral family of the target virus. The comparison of therelative activities of the ON in the target virus and the second virusis accomplished by using the activities of a randomer of the same lengthand chemistry in the both viruses to normalize the IC₅₀ values for theON obtained in the two viruses.

[0403] Thus, if the ON is claimed to have an anti-viral activity againsta certain virus, then the IC₅₀ generation will be determined in thisvirus using one of the assays described herein for the herpesviridae,retroviridae, or paramyxoviridae families, or other assay known in theart. Similarly, IC₅₀ generation will be performed for the ON against asecond virus using one of the assays as described herein for a viruswhose genome has no homology to the sequence of the ON. IC₅₀ generationis also performed for a randomer of equivalent size and chemistryagainst each of the viruses. The IC₅₀ efficacies of the randomer againstthe two viruses are used to normalize the IC₅₀ values for the specificON as follows:

[0404] 1. An algebraic transformation is applied to the IC₅₀ of the ONand the randomer in the first (homologous) virus such that the IC₅₀ ofthe randomer is now 1.

[0405] 2. An algebraic transformation is applied to the IC₅₀ of the ONand the randomer in the second (non-homologous) virus such that the IC₅₀of the randomer in now 1.

[0406] 3. The fold difference in the IC50s for the ON in the homologusversus the non-homologous virus is calculated by dividing thetransformed IC50 of the ON in the non-homologous virus by thetransformed IC50 of the ON in the homologous virus.

[0407] For an ON less than 25 bases in length, the ON shall be deemed tobe acting by a non-sequence complementary mode of action if the folddifference is less than 2. For an ON 25 bases or more in length, the ONshall be deemed to be acting by a non-sequence complimentary mode ofaction if the fold difference in less than 10.

[0408] Test #4: Efficacy in a Different Viral Family

[0409] This test serves to determine if an ON has a drug like activityin a virus where the sequence of said ON is not homologous to anyportion of the viral genome. Thus the ON shall be tested using one ofthe assays described herein for the herpesviridae, retroviridae orparamyxoviridae such that the sequence of the ON tested is nothomologous to any portion of the genome of the virus to be used. An IC₅₀value shall be generated using a minimum of seven concentrations of theON, with three or more points in the linear range. If the resulting doseresponse curve indicates a drug like activity (which can be typically beseen as a decay or sigmoidal curve, having reduced anti-viral efficacywith decreasing concentrations of ON) and the IC₅₀ generated from saidcurve is less than 1 uM, the ON shall be deemed to have a drug likeactivity. If the ON is deemed to have a drug like activity in a virus towhich the ON is not complementary and thus can have no complementarysequence dependent activity, it shall be considered to be acting by anon-sequence complementary mode of action.

[0410] Thresholds Used in These Tests

[0411] There is no scientific or empirical basis, either in the academicor industrial fields, to design an antisense ON which is longer than 25bases. In addition, to our knowledge, there has never been an ONformulation administered in any human trials that used an ON longer than25 nucleotides.

[0412] Given these facts, we have established two different thresholdswhich we use to define a sequence as acting predominantly by anon-sequence complimentary mode of action. For oligos 25 bases and less,the ON must have an antiviral activity which is at least 2-fold greaterthan a randomer or a degenerate ON of the same chemistry in order to beconsidered to be acting predominantly by an antisense mechanism. If anantisense ON is not at least twice as good as a randomer or a degenerateON, then we conclude that more than half of its activity can beattributed to a non-antisense mode of action.

[0413] For ONs 26 bases and larger, the ON must have an antiviralactivity which is at least 10-fold (1 log) greater than a randomer or adegenerate ON of the same chemistry to be considered to be acting by anantisense mechanism. Our rationale for this larger threshold it that,given the current state of the art for ON design for antisense, it isreasonable to assume that oligos that are 26 bases and larger andclaimed to have an antiviral activity were designed with the knowledgeof the invention contained herein (the optimal length of antisense ONsis generally accepted to be between 16 and 21 bases).

[0414] The thresholds described in tests 1 to 3 above are the defaultthresholds. If specifically indicated, other thresholds can be used inthe comparison tests 1 to 3 described above. Thus for example, for ONsunder 25 bases in length and/or ONs 25 or more bases in length, ifspecifically indicated the threshold for determining whether an ON isacting principally by a non-sequence complementary mode of action can beany of 10-fold, 8-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold,1.5-fold, or equal. The threshold described in test 4 above is also adefault threshold. If specifically indicated, the threshold fordetermining whether an ON is acting principally by a non-sequencecomplementary mode of action in test 4 can be an IC₅₀ of less than 1 uM,0.8 uM, 0.6 uM, 0.5 uM, 0.4 uM, 0.3 uM, 0.2 uM or 0.1 uM. Similarly,though the default is that satisfying any one of the above 4 tests issufficient, if specifically indicated, the ON can be required to satisfyany two (e.g., tests 1 & 2, 1 & 3, 1 & 4, 2 & 3, 2& 4 and 3 & 4) or anythree (e.g., tests 1 & 2 & 3, 1 & 3 & 4, and 2 & 3 & 4) or all 4 of thetests at a default threshold, or if specifically indicated, at anotherthreshold as indicated above.

[0415] Antiviral Assay for Herpesviridae

[0416] A plaque reduction assay performed as follows:

[0417] For HSV-1 or HSV-2, VERO cells (ATCC# CCL-81) are grown toconfluence in 12 well tissue culture plates (NUNC or equivalent) at 37deg C. and 5% CO₂ in the presence of MEM supplemented with 10% heatinactivated fetal calf serum and gentamycin, vancomycin and amphoterecinB. Upon reaching confluency, the media is changed to contain 5% fetalcalf serum and antibiotics as detailed above supplemented with eitherHSV-1 (strain KOS, 40-60 PFU total) or HSV-2 (strain MS2, 40-60 PFUtotal). Viral adsorbtion proceeds for 90 minutes, after which cells arewashed and replaced with new “overlay” media containing 5% fetal calfserum and 1% human imunoglobins. Three to four days after adsorbtion,cells are fixed by formalin and plaques are counted following formalinfixation.

[0418] For CMV, human fibroblasts are grown as specified for VERO cellsin the HSV-1/2 assay. Media components and adsorbtion/overlay proceduresare identical with the following exceptions:

[0419] 1. CMV (strain AD169, 40-60 PFU total) is used to infect cellsduring the adsorbtion.

[0420] 2. In the overlay media, 1% human immunoglobins are replaced by4% sea-plaque agarose.

[0421] For other herpesviridae, testing is to be conducted in a plaqueassay described above using an appropriate cellular host and 40-60 PFUof virus during the adsorbtion.

[0422] This test is only valid if identifiable plaques are present inthe absence of compound at the end of the test.

[0423] IC₅₀ is the concentration at which 50% of the plaques are presentcompared to the untreated control.

[0424] Compound to be tested is present during the adsorption and in theoverlay.

[0425] Antiviral Assay for Retroviridae

[0426] Detection of total p24 in the supernatant of HIV-1 infected cellsis performed as follows:

[0427] Human PBMCs are infected with a primary isolate of HIV-1 in thepresence the compound. The cells are then incubated for an additional 7days in fresh medium supplemented with the compound after which thelevels of p24 in the supernatant are measured using a commercial p24ELISA kit (BIOMERIEUX or equivalent.)

[0428] This test is only valid if there is an accumulation of p24 in thetissue culture supernatant in the infected, untreated cells.

[0429] IC₅₀ is the concentration at which the amount of p24 detectableis 50% of the p24 present in the untreated control.

[0430] Compound to be tested is present during the adsorption and in themedia after adsorption.

[0431] Antiviral Assay for Paramyxoviridae

[0432] For RSV, A measurement of CPE is performed as follows:

[0433] Hep2 cells were plated in 96 well plates and allowed to growovernight in MEM plus 5% fetal calf serum at 37 deg C. and 5% CO₂. Thenext day, cells are infected with RSV (strain A2, 10^(8.2)TCID₅₀/ml in100 ul/well) by adsorbtion for 2 hours. Following adsorbtion, media ischanged and after 7 days growth, CPE is measured by conversion of AlamarBlue dye to its fluorescent adduct by living cells.

[0434] This test is only valid if CPE measurement (as measured by AlamarBlue conversion) in infected cells in the absence of compound is 10% ofthe conversion measured in uninfected cells.

[0435] For purposes of IC₅₀ comparison, 100% CPE is set at theconversion level seen in infected cells and 0% CPE is set at theconversion seen in uninfected cells. Therefore IC₅₀ is the concentrationof compound which generates 50% CPE.

[0436] Compound to be tested is present during the adsorption and in themedia after adsorption.

[0437] All patents and other references cited in the specification areindicative of the level of skill of those skilled in the art to whichthe invention pertains, and are incorporated by reference in theirentireties, including any tables and figures, to the same extent as ifeach reference had been incorporated by reference in its entiretyindividually.

[0438] One skilled in the art would readily appreciate that the presentinvention is well adapted to obtain the ends and advantages mentioned,as well as those inherent therein. The methods, variances, andcompositions described herein as presently representative of preferredembodiments are exemplary and are not intended as limitations on thescope of the invention. Changes therein and other uses will occur tothose skilled in the art, which are encompassed within the spirit of theinvention, are defined by the scope of the claims.

[0439] It will be readily apparent to one skilled in the art thatvarying substitutions and modifications may be made to the inventiondisclosed herein without departing from the scope and spirit of theinvention. For example, variations can be made to synthesis conditionsand compositions of the oligonucleotides. Thus, such additionalembodiments are within the scope of the present invention and thefollowing claims.

[0440] The invention illustratively described herein suitably may bepracticed in the absence of any element or elements, limitation orlimitations which is not specifically disclosed herein. Thus, forexample, in each instance herein any of the terms “comprising”,“consisting essentially of” and “consisting of” may be replaced witheither of the other two terms. The terms and expressions which have beenemployed are used as terms of description and not of limitation, andthere is no intention that in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the concepts herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention as defined by theappended claims.

[0441] In addition, where features or aspects of the invention aredescribed in terms of Markush groups or other grouping of alternatives,those skilled in the art will recognize that the invention is alsothereby described in terms of any individual member or subgroup ofmembers of the Markush group or other group.

[0442] Also, unless indicated to the contrary, where various numbericalvalues are provided for embodiments, additional embodiments aredescribed by taking any 2 different values as the endpoints of a range.Such ranges are also within the scope of the described invention.

[0443] Thus, additional embodiments are within the scope of theinvention and within the following claims.

1 36 1 20 DNA Homo sapiens 1 ttgataaata gtactaggac 20 2 22 DNA Humanherpesvirus 1 2 gaagcgttcg cacttcgtcc ca 22 3 16 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 3cttgcggtat tcggaa 16 4 10 DNA Artificial Sequence Description ofArtificial Sequence Synthetic oligonucleotide 4 tccgaagacg 10 5 20 DNAArtificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 5 acacctccga agacgataac 20 6 40 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 6ctacagacat acacctccga agacgataac actagacata 40 7 10 DNA Humanherpesvirus 1 7 cccccatgga 10 8 20 DNA Human herpesvirus 1 8 tacgacccccatggagcccc 20 9 40 DNA Human herpesvirus 1 9 tccagccgca tacgacccccatggagcccc gccccggagc 40 10 21 DNA Artificial Sequence Description ofArtificial Sequence Synthetic oligonucleotide 10 gcgtttgctc ttcttcttgc g21 11 21 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 11 gcgtttgctc ttcttcttgc g 21 12 20 DNAArtificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 12 aaaaaaaaaa aaaaaaaaaa 20 13 20 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 13gggggggggg gggggggggg 20 14 20 DNA Artificial Sequence Description ofArtificial Sequence Synthetic oligonucleotide 14 cccccccccc cccccccccc20 15 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 15 tttttttttt tttttttttt 20 16 20 DNAArtificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 16 acacacacac acacacacac 20 17 20 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 17agagagagag agagagagag 20 18 20 DNA Artificial Sequence Description ofArtificial Sequence Synthetic oligonucleotide 18 tctctctctc tctctctctc20 19 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 19 tgtgtgtgtg tgtgtgtgtg 20 20 40 DNAArtificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 20 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 40 21 40DNA Artificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 21 gggggggggg gggggggggg gggggggggg gggggggggg 40 22 40DNA Artificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 22 cccccccccc cccccccccc cccccccccc cccccccccc 40 23 40DNA Artificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 23 tttttttttt tttttttttt tttttttttt tttttttttt 40 24 40DNA Artificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 24 acacacacac acacacacac acacacacac acacacacac 40 25 40DNA Artificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 25 tctctctctc tctctctctc tctctctctc tctctctctc 40 26 40DNA Artificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 26 agagagagag agagagagag agagagagag agagagagag 40 27 120DNA Artificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 27 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa 60 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa 120 28 120 DNA Artificial Sequence Description ofArtificial Sequence Synthetic oligonucleotide 28 cccccccccc cccccccccccccccccccc cccccccccc cccccccccc cccccccccc 60 cccccccccc cccccccccccccccccccc cccccccccc cccccccccc cccccccccc 120 29 120 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 29gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 60gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 120 30120 DNA Artificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 30 tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt 60 tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt 120 31 240 DNA Artificial Sequence Description ofArtificial Sequence Synthetic oligonucleotide 31 acacacacac acacacacacacacacacac acacacacac acacacacac acacacacac 60 acacacacac acacacacacacacacacac acacacacac acacacacac acacacacac 120 acacacacac acacacacacacacacacac acacacacac acacacacac acacacacac 180 acacacacac acacacacacacacacacac acacacacac acacacacac acacacacac 240 32 240 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 32agagagagag agagagagag agagagagag agagagagag agagagagag agagagagag 60agagagagag agagagagag agagagagag agagagagag agagagagag agagagagag 120agagagagag agagagagag agagagagag agagagagag agagagagag agagagagag 180agagagagag agagagagag agagagagag agagagagag agagagagag agagagagag 240 33240 DNA Artificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 33 atatatatat atatatatat atatatatat atatatatatatatatatat atatatatat 60 atatatatat atatatatat atatatatat atatatatatatatatatat atatatatat 120 atatatatat atatatatat atatatatat atatatatatatatatatat atatatatat 180 atatatatat atatatatat atatatatat atatatatatatatatatat atatatatat 240 34 240 DNA Artificial Sequence Description ofArtificial Sequence Synthetic oligonucleotide 34 cgcgcgcgcg cgcgcgcgcgcgcgcgcgcg cgcgcgcgcg cgcgcgcgcg cgcgcgcgcg 60 cgcgcgcgcg cgcgcgcgcgcgcgcgcgcg cgcgcgcgcg cgcgcgcgcg cgcgcgcgcg 120 cgcgcgcgcg cgcgcgcgcgcgcgcgcgcg cgcgcgcgcg cgcgcgcgcg cgcgcgcgcg 180 cgcgcgcgcg cgcgcgcgcgcgcgcgcgcg cgcgcgcgcg cgcgcgcgcg cgcgcgcgcg 240 35 240 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 35ctctctctct ctctctctct ctctctctct ctctctctct ctctctctct ctctctctct 60ctctctctct ctctctctct ctctctctct ctctctctct ctctctctct ctctctctct 120ctctctctct ctctctctct ctctctctct ctctctctct ctctctctct ctctctctct 180ctctctctct ctctctctct ctctctctct ctctctctct ctctctctct ctctctctct 240 36240 DNA Artificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 36 gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgtgtgtgtgtgt gtgtgtgtgt 60 gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgtgtgtgtgtgt gtgtgtgtgt 120 gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgtgtgtgtgtgt gtgtgtgtgt 180 gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgtgtgtgtgtgt gtgtgtgtgt 240

What is claimed is:
 1. A method for the prophylaxis or treatment of aHSV-1, HSV-2, or CMV infection in a subject, comprising administering toa subject in need of such treatment a therapeutically effective amountof at least one pharmacologically acceptable oligonucleotide at least 29nucleotides in length, wherein the anti-viral activity of saidoligonucleotide occurs principally by a non-sequence complementary modeof action.
 2. The method of claim 1, wherein said subject is a human. 3.An antiviral pharmaceutical composition comprising a therapeuticallyeffective amount of at least one pharmacologically acceptable, antiviraloligonucleotide at least 29 nucleotides in length, wherein saidcomposition is approved for use in humans against HSV-1, HSV-2, or CMV,and the antiviral activity of said oligonucleotide occurs principally bya non-sequence complementary mode of action; and a pharmaceuticallyacceptable carrier.
 4. The antiviral pharmaceutical composition of claim3, adapted for delivery by oral ingestion.
 5. The antiviralpharmaceutical composition of claim 3, adapted for delivery enterally.6. The antiviral pharmaceutical composition of claim 3, adapted fordelivery by injection.
 7. The antiviral pharmaceutical composition ofclaim 3, adapted for delivery by inhalation.
 8. The antiviralpharmaceutical composition of claim 3, adapted for delivery topically.9. The antiviral pharmaceutical composition of claim 3, wherein saidcomposition further comprises a delivery system.
 10. The antiviralpharmaceutical composition of claim 3, wherein said composition furthercomprises a liposomal formulation.
 11. The antiviral pharmaceuticalcomposition of claim 3, wherein said composition further comprises atleast one other antiviral drug in combination.
 12. A kit comprising atleast one anti-viral oligonucleotide or anti-viral oligonucleotideformulation in a labeled package, wherein said oligonucleotide is atleast 29 nucleotides in length, the anti-viral activity of saidoligonucleotide occurs principally by a non-sequence complementary modeof action, and the label on said package indicates that said anti-viraloligonucleotide can be used against HSV-1, HSV-2, or CMV.
 13. The kit ofclaim 12, wherein said kit is approved by a regulatory agency for use inhumans.
 14. The method, pharmaceutical composition, or kit of claim 1,3, or 12, wherein said at least one antiviral oligonucleotide comprisesat least one antiviral randomer oligonucleotide.
 15. The method,pharmaceutical composition, or kit of claim 1, 3, or 12, wherein saidoligonucleotide is not complementary to any portion of the genomicsequence of HSV-1, HSV-2, or CMV.
 16. The method, pharmaceuticalcomposition, or kit of claim 1, 3, or 12, wherein said formulation hasan IC₅₀ for HSV-1, HSV-2, or CMV of 0.10 μM or less.
 17. The method,pharmaceutical composition, or kit of claim 1, 3, or 12, wherein saidoligonucleotide is at least 40 nucleotides in length.
 18. The method,pharmaceutical composition, or kit of claim 1, 3, or 12, wherein eachsaid oligonucleotide comprises at least one modification to its chemicalstructure.
 19. The method, pharmaceutical composition, or kit of claim1, 3, or 12, wherein each said oligonucleotide comprises at least onephosphorothioated linkage.
 20. The method, pharmaceutical composition,or kit of claim 1, 3, or 12, wherein each said oligonucleotide comprisesat least one phosphorothioated linkage and is in a formulationcomprising a delivery system.
 21. The method, pharmaceuticalcomposition, or kit of claim 1, 3, or 12, wherein each saidoligonucleotide comprises at least one 2′-modification to the ribosemoiety.
 22. The method, pharmaceutical composition, or kit of claim 1,3, or 12, wherein each said oligonucleotide comprises at least onemethylphosphonate linkage.
 23. The method, pharmaceutical composition,or kit of claim 1, 3, or 12, wherein each said oligonucleotide comprisesat least one phosphorodithioated linkage.
 24. The method, pharmaceuticalcomposition, or kit of claim 1, 3, or 12, wherein each saidoligonucleotide comprises at least one phosphorodithioated linkage andis in a formulation comprising a delivery system.
 25. The method,pharmaceutical composition, or kit of claim 1, 3, or 12, wherein saidoligonucleotide is a concatemer consisting of two or moreoligonucleotide sequences joined by a linker.
 26. The method,pharmaceutical composition, or kit of claim 1, 3, or 12, wherein saidoligonucleotide is linked or conjugated at one or more nucleotideresidues, to a molecule modifying the characteristics of theoligonucleotide to obtain one or more characteristics selected from thegroup consisting of higher stability, lower serum interaction, highercellular uptake, higher viral protein interaction, an improved abilityto be formulated for delivery, a detectable signal, higher antiviralactivity, better pharmacokinetic properties, specific tissuedistribution, lower toxicity.
 27. The method, pharmaceuticalcomposition, or kit of claim 1, 3, or 12, wherein said oligonucleotideis double stranded.
 28. The method, pharmaceutical composition, or kitof claim 1, 3, or 12, wherein said oligonucleotide binds to one or moreviral components.
 29. The method, pharmaceutical composition, or kit ofclaim 1, 3, or 12, wherein at least a portion of the sequence of saidoligonucleotide is derived from a viral genome.
 30. The method,pharmaceutical composition, or kit of claim 1, 3, or 12, comprising amixture of at least two different antiviral oligonucleotides.
 31. Themethod, pharmaceutical composition, or kit of claim 30, wherein aplurality of said different oligonucleotides are at least 29 nucleotidesin length.
 32. The method, pharmaceutical composition, or kit of claim30, wherein a plurality of said different oligonucleotides are at least40 nucleotides in length.
 33. A method for selecting an antiviraloligonucleotide for use as an anti-viral agent, comprising synthesizinga plurality of different oligonucleotides, wherein at least one of saiddifferent oligonucleotides is at least 29 nucleotides in length; testingsaid oligonucleotides for activity in inhibiting the ability of HSV-1,HSV-2, or CMV to produce infectious virions, selecting a saidoligonucleotide having a pharmaceutically acceptable level of activityfor use as an anti-viral agent.
 34. The method of claim 33, wherein saiddifferent oligonucleotides comprise randomers of different lengths. 35.The method of claim 33, wherein said different oligonucleotides comprisea set of oligonucleotides of different length, each oligonucleotide insaid set comprising the sequence of the shortest oligonucleotide in saidset.
 36. The method of claim 33, wherein said different oligonucleotidescomprise a plurality of oligonucleotides comprising a randomer segmentat least 6 nucleotides in length.
 37. The method of claim 33, whereinsaid different oligonucleotides are not complementary to any mRNAsequence of HSV-1, HSV-2, or CMV.
 38. A method for the prophylaxis ortreatment of a HSV-1, HSV-2, or CMV infection in a subject, comprisingadministering to a subject in need of such treatment a therapeuticallyeffective amount of at least one pharmacologically acceptableoligonucleotide randomer at least 10 nucleotides in length, wherein theanti-viral activity of said randomer occurs principally by anon-sequence complementary mode of action.